Peptides effective in the treatment of tumors and other conditions requiring the removal or destruction of cells

ABSTRACT

The invention is directed to methods of treating conditions requiring removal or destruction of harmful or unwanted cells in a patient, such as benign and malignant tumors, using compounds containing or based on peptides comprising a part of the amino acid sequence of a neural thread protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of US application Ser. No.12/171,462, filed on Jul. 11, 2008, now U.S. Pat. No. 8,293,703, whichis a Divisional of U.S. application Ser. No. 10/920,313, filed Oct. 12,2004, now U.S. Pat. No. 7,408,021, which is a Continuation of U.S.application Ser. No. 10/294,891, filed Nov. 15, 2002, now U.S. Pat. No.7,317,077, which claims priority under 35 USC 119(e) to U.S. ProvisionalApplication No. 60/331,477, filed on Nov. 16, 2001, the contents ofwhich are incorporated herein in their entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to methods of treating conditionsrequiring removal or destruction of cellular elements, such as benign ormalignant tumors in humans, using compounds based on peptides comprisingamino acid sequences corresponding to, similar to or homologous to partof the amino acid sequence of neural thread proteins. The methodincludes, but is not limited to, administering the compoundsintramuscularly, orally, intravenously, intrathecally, intratumorally,intranasally, topically, transdermally, etc., either alone or conjugatedto a carrier.

2. Description of Related Art

The essence of many medical treatments and procedures involves theremoval or destruction of harmful or unwanted tissue. Examples of suchimportant treatments include the surgical removal of cancerous growths,the destruction of metatastic tumors through chemotherapy, and thereduction of glandular (e.g. prostate) hyperplasia. Other examplesinclude the removal of unwanted facial hair, the removal of warts, andthe removal of unwanted fatty tissue.

There is an obvious need for an effective agent that will destroy andhence either facilitate the removal of or inhibit the further growth ofharmful or unwanted cells and tissue but will have mainly local effectsand minimal or absent systemic toxicity.

Neural thread proteins and their related molecules are one class of suchagents, as disclosed in pending U.S. patent application Ser. No.10/092,934, entitled: Methods of Treating Tumors and Related ConditionsUsing Neural Thread Proteins, the disclosure of which is incorporated bereference herein in its entirety. Certain fragments of neural threadproteins and related proteins are disclosed as useful in treating tumorsand other conditions requiring removal or destruction of cells in U.S.patent application Ser. No. 10/153,334, entitled: Peptides Effective InThe Treatment Of Tumors And Other Conditions Requiring The Removal OrDestruction Of Cells; Ser. No. 10/198,069, entitled: Peptides EffectiveIn The Treatment Of Tumors And Other Conditions Requiring The Removal OrDestruction Of Cells; and Ser. No. 10/198,070, entitled: PeptidesEffective In The Treatment Of Tumors And Other Conditions Requiring TheRemoval Or Destruction Of Cells, the disclosures of each of which areincorporated by reference herein in their entirety.

Disclosed herein are certain other fragments of neural thread proteinsthat also are useful in treating tumors and other conditions requiringremoval or destruction of cells.

Cancer is an abnormality in a cell's internal regulatory mechanisms thatresults in uncontrolled growth and reproduction of the cell. Normalcells make up tissues, and when these cells lose their ability to behaveas a specified, controlled, and coordinated unit, (dedifferentiation),the defect leads to disarray amongst the cell population. When thisoccurs, a tumor is formed.

Benign overgrowths of tissue are abnormalities in which it is desirableto remove cells from an organism. Benign tumors are cellularproliferations that do not metastasize throughout the body but do,however, cause disease symptoms. Such tumors can be lethal if they arelocated in inaccessible areas in organs such as the brain. There arebenign tumors of organs including lung, brain, skin, pituitary, thyroid,adrenal cortex and medulla, ovary, uterus, testis, connective tissue,muscle, intestines, ear, nose, throat, tonsils, mouth, liver, gallbladder, pancreas, prostate, heart, and other organs.

Surgery often is the first step in the treatment of cancer. Theobjective of surgery varies. Sometimes it is used to remove as much ofthe evident tumor as possible, or at least to “debulk” it (remove themajor bulk(s) of tumor so that there is less that needs to be treated byother means). Depending on the cancer type and location, surgery mayalso provide some symptomatic relief to the patient. For instance, if asurgeon can remove a large portion of an expanding brain tumor, thepressure inside the skull will decrease, leading to improvement in thepatient's symptoms.

Not all tumors are amenable to surgery. Some may be located in parts ofthe body that make them impossible to completely remove. Examples ofthese would be tumors in the brainstem (a part of the brain thatcontrols breathing) or a tumor which has grown in and around a majorblood vessel. In these cases, the role of surgery is limited due to thehigh risk associated with tumor removal.

In some cases, surgery is not used to debulk tumor because it is simplynot necessary. An example is Hodgkin's lymphoma, a cancer of the lymphnodes that responds very well to combinations of chemotherapy andradiation therapy. In Hodgkin's lymphoma, surgery is rarely needed toachieve cure, but almost always used to establish a diagnosis.

Chemotherapy is another common form of cancer treatment. Essentially, itinvolves the use of medications (usually given by mouth or injection)which specifically attack rapidly dividing cells (such as those found ina tumor) throughout the body. This makes chemotherapy useful in treatingcancers that have already metastasized, as well as tumors that have ahigh chance of spreading through the blood and lymphatic systems but arenot evident beyond the primary tumor. Chemotherapy may also be used toenhance the response of localized tumors to surgery and radiationtherapy. This is the case, for example, for some cancers of the head andneck.

Unfortunately, other cells in the human body that also normally dividerapidly (such as the lining of the stomach and hair) also are affectedby chemotherapy. For this reason, many chemotherapy agents induceundesirable side effects such as nausea, vomiting, anemia, hair loss orother symptoms. These side effects are temporary, and there existmedications that can help alleviate many of these side effects. As ourknowledge has continued to grow, researchers have devised newerchemotherapeutic agents that are not only better at killing cancercells, but that also have fewer side effects for the patient.

Chemotherapy is administered to patients in a variety of ways. Someinclude pills and others are administered by an intravenous or otherinjection. For injectable chemotherapy, a patient goes to the doctor'soffice or hospital for treatment. Other chemotherapeutic agents requirecontinuous infusion into the bloodstream, 24 hours a day. For thesetypes of chemotherapy, a minor surgical procedure is performed toimplant a small pump worn by the patient. The pump then slowlyadministers the medication. In many cases, a permanent port is placed ina patient's vein to eliminate the requirement of repeated needle sticks.

Radiation therapy is another commonly used weapon in the fight againstcancer. Radiation kills cancer by damaging the DNA within the tumorcells. The radiation is delivered in different ways. The most commoninvolves pointing a beam of radiation at the patient in a highly precisemanner, focusing on the tumor. To do this, a patient lies on a table andthe beam moves around him/her. The procedure lasts minutes, but may bedone daily for several weeks (depending on the type of tumor), toachieve a particular total prescribed dose.

Another radiation method sometimes employed, called brachytherapy,involves taking radioactive pellets (seeds) or wires and implanting themin the body in the area of the tumor. The implants can be temporary orpermanent. For permanent implants, the radiation in the seeds decaysover a period of days or weeks so that the patient is not radioactive.For temporary implants, the entire dose of radiation is usuallydelivered in a few days, and the patient must remain in the hospitalduring that time. For both types of brachytherapy, radiation isgenerally delivered to a very targeted area to gain local control over acancer (as opposed to treating the whole body, as chemotherapy does.)

Some highly selected patients may be referred for bone marrowtransplants. This procedure usually is performed either because apatient has a cancer that is particularly aggressive or because theyhave a cancer that has relapsed after being treated with conventionaltherapy. Bone marrow transplantation is a complicated procedure. Thereare many types, and they vary in their potential for causing sideeffects and cure. Most transplants are performed at special centers, andin many cases, their use is considered investigational.

A number of other therapies exist, although most of them are still beingexplored in clinical trials and have not yet become standard care.Examples include the use of immunotherapy, monoclonal antibodies,anti-angiogenesis factors and gene therapy.

Immunotherapy: There are various techniques designed to help thepatient's own immune system fight the cancer, quite separately fromradiation or chemotherapy. Oftentimes, to achieve the goal researchersinject the patient with a specially derived vaccine.

Monoclonal Antibodies: These are antibodies designed to attach tocancerous cells (and not normal cells) by taking advantage ofdifferences between cancerous and non-cancerous cells in their anitgenicand/or other characteristics. The antibodies can be administered to thepatient alone or conjugated to various cytotoxic compounds or inradioactive form, such that the antibody preferentially targets thecancerous cells, thereby delivering the toxic agent or radioactivity tothe desired cells.

Anti-Angiogenesis Factors: As cancer cells rapidly divide and tumorsgrow, they can soon outgrow their blood supply. To compensate for this,some tumors secrete a substance believed to help induce the growth ofblood vessels in their vicinity, thus providing the cancer cells with avascular source of nutrients. Experimental therapies have been designedto arrest the growth of blood vessels to tumors.

Gene Therapy: Cancer is the product of a series of mutations thatultimately lead to the production of a cancer cell and its excessiveproliferation. Cancers can be treated by introducing genes to the cancercells that will act either to check or stop the cancer's proliferation,turn on the cell's programmed cell mechanisms to destroy the cell,enhance immune recognition of the cell, or express a pro-drug thatconverts to a toxic metabolite or a cytokine that inhibits tumor growth.

Benign tumors and malformations also can be treated by a variety ofmethods including surgery, radiotherapy, drug therapy, thermal orelectric ablation, cryotherapy, and others. Although benign tumors donot metastasize, they can grow large and they can recur. Surgicalextirpation of benign tumors has all the difficulties and side effectsof surgery in general and oftentimes must be repeatedly performed forsome benign tumors, such as for pituitary adenomas, meningeomas of thebrain, prostatic hyperplasia, and others.

Other conditions involving unwanted cellular elements exist whereselective cellular removal is desirable. For example, heart disease andstrokes commonly are caused by atherosclerosis, which is a proliferativelesion of fibrofatty and modified smooth muscle elements that distortthe blood vessel wall, narrow the lumen, constrict blood flow,predispose to focal blood clots, and ultimately lead to blockage andinfarction. There are various treatments for atherosclerosis such asbypass grafts; artificial grafts; angioplasty with recanalization,curettage, radiation, laser, or other removal; pharmacotherapy toinhibit atherosclerosis through lipid reduction; anti-clottingtherapies; and general measures of diet, exercise, and lifestyle. Amethod for removing atherosclerotic lesions without the risk and sideeffects of surgical procedures is needed.

Other examples of unwanted cellular elements where selective cellularremoval is desirable include viral induced growths, such as warts.Another example is hypertrophic inflammatory masses found ininflammatory conditions, and hypertrophic scars or keloids. Still otherexamples are found in cosmetic contexts such as the removal of unwantedhair, e.g., facial hair, or for shrinkage of unwanted tissue areas forcosmetic purposes, such as in the facial dermis and connective tissuesor in the dermas and connective tissue of the extremities.

Other examples of unwanted cellular elements where selective cellularremoval or the inhibition of cellular proliferation is desirable includestenosis and restenosis of any artery, valve or canal in the circulatorysystem including, but not limited to, valves (e.g., aortic stenosiswhich involves narrowing of the aortic valve orifice), coronary arteries(e.g., coronary ostial sclerosis which involves narrowing of the mouthsof the coronary arteries), carotid arteries, and renal arteries. Otherexamples include the inhibition or removal of unwanted cellular growthor accumulation causing the partial or complete occulsion of medicaldevices such as stents placed or implanted within a blood vessel fortreating stenoses, strictures or aneurysms therein or within the urinarytract and in bile ducts.

Still other examples will be obvious to those of ordinary skill in theart. In all or most of these examples there is a need for treatmentsthat can remove or destroy the unwanted cellular elements without therisks and side effects of conventional therapies or remove the unwantedcellular elements with more precision.

Neural thread proteins (NTP) are a family of recently characterizedbrain proteins. One member of this family, AD7c-NTP, is a ˜41 kDmembrane associated phosphoprotein with functions associated withneuritic sprouting (de la Monte et al., J. Clin. Invest., 100:3093-3104(1997); de la Monte et al., Alz. Rep., 2:327-332 (1999); de la Monte S Mand Wands J R, Journal of Alzheimer's Disease, 3:345-353 (2001)). Thegene that encodes AD7c-NTP and predicted protein sequence for AD7c-NTPhas been identified and described (de la Monte et al., J. Clin. Invest.,100:3093-3104 (1997)). In addition to the ˜41 kD species, other speciesof neural thread protein (˜26 kD, ˜21 kD, ˜17 kD, and ˜15 kD) have beenidentified and associated with neuroectodermal tumors, astrocytomas, andglioblastomas and with injury due to hypoxia, schema, or cerebralinfarction (Xu et al., Cancer Research, 53:3823-3829 (1993); de la Monteet al., J. Neuropathol. Exp. Neurol., 55(10):1038-50 (1996), de la Monteet al., J. Neurol. Sci., 138(1-2):26-35 (1996); de la Monte et al., J.Neurol. Sci., 135(2):118-25 (1996); de la Monte et al., J. Clin.Invest., 100:3093-3104 (1997); and de la Monte et al., Alz. Rep.,2:327-332 (1999)).

Species of neural thread protein have been described and claimed in U.S.Pat. Nos. 5,948,634; 5,948,888; and 5,830,670, all for “Neural ThreadProtein Gene Expression and Detection of Alzheimer's Disease” and inU.S. Pat. No. 6,071,705 for “Method of Detecting Neurological Disease orDysfunction.” The disclosures of these patents are specificallyincorporated herein by reference in their entirety. As describedtherein, NTP is upregulated and produced during cell death. Thus, deadand dying nerve cells are described as overproducing NTP, andaccordingly, its presence indicates the death of nerve cells and theonset of Alzheimer's disease (AD).

Other species of neural thread protein have been identified as otherproducts of the AD7c-NTP gene (e.g. a 112 amino acid protein describedin NCBI Entrez-Protein database Accession #XP_(—)032307 PID g15928971)or as being similar to neural thread proteins (e.g. a 106 amino acidprotein described in NCBI Entrez-Protein database Accession #AAH14951PID g15928971, and a 61 amino acid protein described in NCBIEntrez-Protein database Accession #AAH02534 PID g12803421).

Neural thread protein is associated with AD and NTP is upregulated inassociation with cell death in AD. AD7c-NTP mRNA is upregulated in ADbrain compared to controls; AD7c-NTP protein levels in brain and in CSFare higher in AD than controls; and AD7c-NTP immunoreactivity is foundin senile plaques, in neurofibrillary tangles (NFT), in degeneratingneurons, neuropil threads, and dystrophic neurotic sprouts in AD andDown syndrome brains (Ozturk et al., Proc. Natl. Acad. Sci. USA,86:419-423 (1989); de la Monte et al., J. Clin. Invest., 86(3):1004-13(1990); de la Monte et al., J. Neurol. Sci., 113(2):152-64 (1992); de laMonte et al., Ann. Neurol., 32(6):733-42 (1992); de la Monte et al., J.Neuropathol. Exp. Neurol., 55(10):1038-50 (1996), de la Monte et al., J.Neural. Sci., 138(1-2):26-35 (1996); de la Monte et al., J. Neurol.Sci., 135(2):118-25 (1996); de la Monte et al., J. Clin. Invest.,100:3093-3104 (1997); and de la Monte et al., Alz. Rep., 2:327-332(1999)). NTP is localized within cells, within fine processes within theneuropil, or is extracellular in both AD and Down's Syndrome brains. dela Monte et al., Ann. Neurol., 32(6):733-42 (1992).

Elevated levels of AD7c-NTP protein have been found in both CSF andurine of AD patients (de la Monte and Wands, Front Biosci 7: 989-96(2002); de la Monte and Wands, Journal of Alzheimer's Disease 3: 345-353(2001); Munzar et al, Alzheimer's Reports 4: 61-65 (2001); Kahle et al,Neurology 54: 1498-1504 (2000); Munzar et al, Alzheimer Reports 3:155-159 (2000); de la Monte et al, Alzheimer's Reports 2: 327-332(1999); and de la Monte et al, J Clin Invest 100: 3093-3104 (1997).

Over-expression of NTP also has been linked to the process of cell deathin Alzheimer's disease (de la Monte and Wands, J. Neuropathol. Exp.Neurol., 60:195-207 (2001); de la Monte and Wands, Cell Mol Life Sci 58:844-49 (2001). AD7c-NTP has also been identified in Down's Syndromebrain tissue (Wands et al., International Patent Publication No. WO90/06993; de la Monte et al, J Neurol Sci 135: 118-25 (1996); de laMonte et al., Alz. Rep., 2:327-332 (1999)). There is some evidence thatover-expression of NTP also may be associated with normal tensionglaucoma (Golubnitschaja-Labudova et al, Curr Eye Res 21: 867-76(2000)).

NTP has proven to be an effective agent for causing cell death both invitro in glioma and neuroblastoma cell cultures and in vivo in normalrodent muscle tissue, subcutaneous connective tissue, and dermis, and ina variety of different human and non-human origin tumors, includingmammary carcinoma, skin carcinoma and papilloma, colon carcinoma, gliomaof brain, and others in rodent models. See the pending U.S. patentapplication Ser. No. 10/092,934, Methods of Treating Tumors and RelatedConditions Using Neural Thread Proteins.

Certain peptide sequences and fragments of AD7c-NTP and other species ofNTP also have proven to be effective agents for causing cell death bothin vitro in glioma and neuroblastoma cell cultures and/or in vivo innormal rodent muscle tissue, subcutaneous connective tissue, dermis andother tissue. See U.S. patent application Ser. No. 10/153,334, entitled:Peptides Effective In The Treatment Of Tumors And Other ConditionsRequiring The Removal Or Destruction Of Cells; Ser. No. 10/198,069,entitled: Peptides Effective In The Treatment Of Tumors And OtherConditions Requiring The Removal Or Destruction Of Cells; and Ser. No.10/198,070, entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells, thedisclosures of each of which are incorporated by reference herein intheir entirety.

Throughout this description, including the foregoing description ofrelated art, any and all publicly available documents described herein,including any and all U.S. patents, are specifically incorporated byreference herein in their entirety. The foregoing description of relatedart is not intended in any way as an admission that any of the documentsdescribed therein, including pending United States patent applications,are prior art to the present invention. Moreover, the description hereinof any disadvantages associated with the described products, methods,and/or apparatus, is not intended to limit the invention. Indeed,aspects of the invention may include certain features of the describedproducts, methods, and/or apparatus without suffering from theirdescribed disadvantages.

There remains a need in the art for new, less toxic treatments fortreating unwanted cellular elements. The present invention satisfiesthese needs.

SUMMARY OF THE INVENTION

This invention is premised in part on the discovery that peptidescontaining amino acid sequences corresponding to part of the amino acidsequences of other species of neural thread proteins other than AD7c-NTPare capable of treating and/or killing unwanted cellular proliferations.These unwanted cellular proliferations include, inter alia, benign andmalignant tumors, glandular (e.g. prostate) hyperplasia, unwanted facialhair, warts, and unwanted fatty tissue.

The present invention is directed to methods of treating unwantedcellular proliferations, (benign and malignant tumors, glandular (e.g.prostate) hyperplasia, unwanted facial hair, warts, and unwanted fattytissue) comprising administering to a mammal in need thereof atherapeutically effective amount of a peptide comprising an amino acidsequence (or more than one sequence) corresponding to part of the aminoacid sequence of a species of neural thread protein (NTP) other thanAD7c-NTP.

Such a peptide (“NTP peptide”) can be administered alone or conjugatedto a carrier or an antibody. The NTP peptide can be administeredintramuscularly, orally, intravenously, intraperitoneally,intracerebrally (intraparenchymally), intracerebroventricularly,intratumorally, intralesionally, intradermally, intrathecally,intranasally, intraocularly, intraarterially, topically, transdermally,via an aerosol, infusion, bolus injection, implantation device,sustained release system etc., either alone or conjugated to a carrier.Alternatively, the NTP peptide can be expressed in vivo by administeringa gene that expresses the peptide, by administering a vaccine thatinduces such production or by introducing cells, bacteria or virusesthat express the peptide in vivo, because of genetic modification orotherwise.

In addition, the NTP peptide may be used in conjunction with othertherapies for treating benign and malignant tumors and other unwanted orharmful cellular growths.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory and are intended to providefurther explanation of the invention as claimed. Other objects,advantages, and features will be readily apparent to those skilled inthe art from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the complete amino acid sequences of the 122 amino acidneural thread protein (Sequence 40 from U.S. Pat. Nos. 5,830,670,5,948,634, and 5,948,888; NCBI Entrez-Protein Accession #AAE25447 PIDg10048540) [SEQ ID NO. 1].

FIG. 2: Shows the complete amino acid sequences of the 112 amino acidneural thread protein (NCBI Entrez-Protein Accession #XP_(—)032307 PIDg15928971) [SEQ ID NO. 2].

FIG. 3: Shows the complete amino acid sequences of a 106 amino acidneural thread protein-like protein (NCBI Entrez-Protein Accession#AAH14951 PID g15928971) [SEQ ID NO. 3].

FIG. 4: Shows the complete amino acid sequences of the 98 amino acidneural thread protein (Sequence 30 from U.S. Pat. Nos. 5,830,670,5,948,634, and 5,948,888; NCBI Entrez-Protein Accession #AAE25445, PIDg10048538) [SEQ ID NO. 4].

FIG. 5: Shows the complete amino acid sequences of the 75 amino acidneural thread protein (Sequence 48 from U.S. Pat. Nos. 5,830,670,5,948,634, and 5,948,888; NCBI Entrez-Protein Accession #AAE25448, PIDg10048541) [SEQ ID NO. 5].

FIG. 6: Shows the complete amino acid sequences of the 68 amino acidneural thread protein (Sequence 36 from U.S. Pat. Nos. 5,830,670,5,948,634, and 5,948,888; NCBI Entrez-Protein Accession #AAE25446, PIDg10048539) [SEQ ID NO. 6].

FIG. 7: Shows the complete amino acid sequences of the 61 amino acidneural thread protein-like protein (NCBI Entrez-Protein Accession#AAH02534, PID g12803421) [SEQ ID NO. 7].

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present proteins, nucleotide sequences, peptides, etc., andmethods are described, it is understood that this invention is notlimited to the particular methodology, protocols, cell lines, vectors,and reagents described, as these may vary. It also is to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

Terms and phrases used herein are defined as set forth below unlessotherwise specified.

Throughout this description, the singular forms “a,” “an,” and “the”include plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a host cell” includes a plurality ofsuch host cells, and a reference to “an antibody” is a reference to oneor more antibodies and equivalents thereof known to those skilled in theart, and so forth.

The term “AD7c-NTP” refers to the ˜41 kD protein and the gene and thenucleic acid sequences coding for it described in de la Monte et al., J.Clin. Invest., 100:3093-104 (1997), in Sequences 120 and 121 of U.S.Pat. Nos. 5,948,634; 5,948,888; and 5,830,670.

The term “NTP” refers to neural thread proteins and related molecules(including pancreatic thread protein) other than AD7c-NTP as describedin U.S. Pat. Nos. 5,948,634; 5,948,888; 5,830,670 and 6,071,705 and inde la Monte et al., J. Neuropathol. Exp. Neurol., 55(10):1038-50 (1996),de la Monte et al., J. Neurol. Sci., 138(1-2):26-35 (1996); de la Monteet al., J. Neurol. Sci., 135(2):118-25 (1996), de la Monte et al., J.Clin. Invest., 100:3093-3104 (1997) and de la Monte et al., Alz. Rep.,2:327-332 (1999). The term “NTP” includes, but is not limited:

-   -   (a) the ˜42, ˜26, ˜21, ˜17, ˜14, and ˜8 kD species of neural        thread protein as described in U.S. Pat. Nos. 5,948,634;        5,948,888; 5,830,670 and 6,071,705 and in de la Monte et al., J.        Neuropathol. Exp. Neurol., 55(10):1038-50 (1996), de la Monte et        al., J. Neurol. Sci., 138(1-2):26-35 (1996); de la Monte et        al., J. Neurol. Sci., 135(2):118-25 (1996), de la Monte et        al., J. Clin. Invest., 100:3093-3104 (1997) and de la Monte et        al., Alz. Rep., 2:327-332 (1999);    -   (b) proteins specifically recognized by monoclonal antibody #2        on deposit with the American Type Culture Collection, Manassas,        Va., under accession number HB-12546 or monoclonal antibody #5        on deposit with the American Type Culture Collection, Manassas,        Va., under accession number HB-12545;    -   (c) proteins coded by the AD7c-NTP gene, including splice        variants;    -   (d) the 122 amino acid neural thread protein described in        Sequence 40 from U.S. Pat. Nos. 5,830,670; 5,948,634 and        5,948,888 and listed in NCBI Entrez-Protein Accession #AAE25447,        PID g10048540, the amino acid sequences for which is illustrated        in FIG. 1 (“NTP[122]”);    -   (e) the 112 amino acid neural thread protein listed in NCBI        Entrez-Protein Accession #XP_(—)032307, PID g14725132, the amino        acid sequences for which is illustrated in FIG. 2 (“NTP[112]”);    -   (f) a 106 amino acid neural thread protein-like protein listed        in NCBI Entrez-Protein Accession #AAH14951 PID g15928971, the        amino acid sequences for which is illustrated in FIG. 3        (“NTP[106]”);    -   (g) the 98 amino acid neural thread protein described in        Sequence 30 from U.S. Pat. Nos. 5,830,670; 5,948,634 and        5,948,888 and listed in NCBI Entrez-Protein Accession #AAE25445,        PID g10048538, the amino acid sequences for which is illustrated        in FIG. 4 (“NTP[98]”);    -   (h) the 75 amino acid neural thread protein described in        Sequence 48 from U.S. Pat. Nos. 5,830,670; 5,948,634 and        5,948,888 and listed in NCBI Entrez-Protein Accession #AAE25448,        PID g10048541, the amino acid sequences for which is illustrated        in FIG. 5 (“NTP[75]”);    -   (i) the 68 amino acid neural thread protein described in        Sequence 36 from U.S. Pat. Nos. 5,830,670; 5,948,634 and        5,948,888 and listed in NCBI Entrez-Protein Accession #AAE25446,        PID g10048539, the amino acid sequences for which is illustrated        in FIG. 6 (“NTP[68]”);    -   (j) the 61 amino acid neural thread protein-like protein listed        in NCBI Entrez-Protein Accession #AAH02534, PID g12803421, the        amino acid sequences for which is illustrated in FIG. 7        (“NTP[61]”);    -   (k) pancreatic thread protein;    -   (l) the neural pancreatic thread protein (nPTP) described in        U.S. Pat. No. 6,071,705; and    -   (m) proteins specifically recognized by the antibodies produced        by a hybridoma from the group consisting of HB 9934, HB 9935,        and HB 9936 deposited at the American Type Culture Collection.        The term “NTP” includes homologues, fragments, derivatives,        variants, fusion proteins, and peptide mimetics of NTP proteins        unless the context indicates otherwise.

The expression “NTP peptide” refers to peptides comprising amino acidsequences corresponding to at least a part of the amino acid sequence ofNTP, of a species of NTP, or to fragments of a species of NTP andincludes homologues, fragments, derivatives, variants, fusion proteins,and peptide mimetics of such peptides unless the context indicatesotherwise.

The term “fragment” refers to a protein or polypeptide that consists ofa continuous subsequence of the amino acid sequence of an NTP protein orNTP peptide and includes naturally occurring fragments such as splicevariants and fragments resulting from naturally occurring in vivoprotease activity. Such a fragment may be truncated at the aminoterminus, the carboxy terminus, and/or internally (such as by naturalsplicing). Such fragments may be prepared with or without an aminoterminal methionine. The term “fragment” includes fragments, whetheridentical or different, from the same NTP protein or NTP peptide, with acontiguous amino acid sequence in common or not, joined together, eitherdirectly or through a linker.

The term “variant” refers to a protein or polypeptide in which one ormore amino acid substitutions, deletions, and/or insertions are presentas compared to the amino acid sequence of an NTP protein or NTP peptideand includes naturally occurring allelic variants or alternative splicevariants of an NTP protein or NTP peptide. The term “variant” includesthe replacement of one or more amino acids in a peptide sequence with asimilar or homologous amino acid(s) or a dissimilar amino acid(s). Thereare many scales on which amino acids can be ranked as similar orhomologous. (Gunnar von Heijne, Sequence Analysis in Molecular Biology,p. 123-39 (Academic Press, New York, N.Y. 1987.) Preferred variantsinclude alanine substitutions at one or more of amino acid positions.Other preferred substitutions include conservative substitutions thathave little or no effect on the overall net charge, polarity, orhydrophobicity of the protein. Conservative substitutions are set forthin Table 2 below.

TABLE 2 Conservative Amino Acid Substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Uncharged Polar: glutamineasparagine serine threonine tyrosine Non-Polar: phenylalanine tryptophancysteine glycine alanine valine proline methionine leucine isoleucine

Table 3 sets out another scheme of amino acid substitution:

TABLE 3 Original Residue Substitutions Ala gly; ser Arg lys Asn gln; hisAsp glu Cys ser Gln asn Glu asp Gly ala; pro His asn; gln Ile leu; valLeu ile; val Lys arg; gln; glu Met leu; tyr; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Other variants can consist of less conservative amino acidsubstitutions, such as selecting residues that differ more significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionsthat in general are expected to have a more significant effect onfunction are those in which (a) glycine and/or proline is substituted byanother amino acid or is deleted or inserted; (b) a hydrophilic residue,e.g., seryl or threonyl, is substituted for (or by) a hydrophobicresidue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) acysteine residue is substituted for (or by) any other residue; (d) aresidue having an electropositive side chain, e.g., lysyl, arginyl, orhistidyl, is substituted for (or by) a residue having an electronegativecharge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky sidechain, e.g., phenylalanine, is substituted for (or by) one not havingsuch a side chain, e.g., glycine. Other variants include those designedto either generate a novel glycosylation and/or phosphorylation site(s),or those designed to delete an existing glycosylation and/orphosphorylation site(s). Variants include at least one amino acidsubstitution at a glycosylation site, a proteolytic cleavage site and/ora cysteine residue. Variants also include NTP proteins and NTP peptideswith additional amino acid residues before or after the NTP protein orNTP peptide amino acid sequence on linker peptides. For example, acysteine residue may be added at both the amino and carboxy terminals ofan NTP Peptide in order to allow the cyclisation of the NTP Peptide bythe formation of a di-sulphide bond. The term “variant” also encompassespolypeptides that have the amino acid sequence of an NTP peptide with atleast one and up to 25 or more additional amino acids flanking eitherthe 3′ or 5′ end of the NTP peptide.

The term “derivative” refers to a chemically modified protein orpolypeptide that has been chemically modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques, as forexample, by addition of one or more polyethylene glycol molecules,sugars, phosphates, and/or other such molecules, where the molecule ormolecules are not naturally attached to wild-type NTP proteins or NTPPeptides. Derivatives include salts. Such chemical modifications arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and they are well known to thoseof skill in the art. It will be appreciated that the same type ofmodification may be present in the same or varying degree at severalsites in a given protein or polypeptide. Also, a given protein orpolypeptide may contain many types of modifications. Modifications canoccur anywhere in a protein or polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, Proteins—Structure And Molecular Properties, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993) and Wold, F.,“Posttranslational Protein Modifications Perspectives and Prospects,”pgs. 1-12 in Posttranslational Covalent Modification Of Proteins, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., “Protein Synthesis:Posttranslational Modifications and Aging,” Ann. N.Y. Acad. Sci. 663:48-62 (1992). The term “derivatives” include chemical modificationsresulting in the protein or polypeptide becoming branched or cyclic,with or without branching. Cyclic, branched and branched circularproteins or polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

The term “homologue” refers to a protein that is at least 60 percentidentical in its amino acid sequence of an NTP protein or NTP peptide,as the case may be, as determined by standard methods that are commonlyused to compare the similarity in position of the amino acids of twopolypeptides. The degree of similarity or identity between two proteinscan be readily calculated by known methods, including but not limited tothose described in Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo H. and Lipman, D., SIAM, J. Applied Math.,48: 1073 (1988). Preferred methods to determine identity are designed togive the largest match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs.

Preferred computer program methods useful in determining the identityand similarity between two sequences include, but are not limited to,the GCG program package (Devereux, J., et al., Nucleic Acids Research,12(1): 387 (1984)), BLASTP, BLASTN, and FASTA, Atschul, S. F. et al., J.Molec. Biol., 215: 403410 (1990). The BLAST X program is publiclyavailable from NCBI and other sources (BLAST Manual, Altschul, S., etal., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol.Biol., 215: 403-410 (1990). By way of example, using a computeralgorithm such as GAP (Genetic Computer Group, University of Wisconsin,Madison, Wis.), the two proteins or polypeptides for which the percentsequence identity is to be determined are aligned for optimal matchingof their respective amino acids (the “matched span”, as determined bythe algorithm).

A gap opening penalty (which is calculated as 3×(times) the averagediagonal; the “average diagonal” is the average of the diagonal of thecomparison matrix being used; the “diagonal” is the score or numberassigned to each perfect amino acid match by the particular comparisonmatrix) and a gap extension penalty (which is usually 1/10 times the gapopening penalty), as well as a comparison matrix such as PAM 250 orBLOSUM 62 are used in conjunction with the algorithm. A standardcomparison matrix (see Dayhoff et al. in: Atlas of Protein Sequence andStructure, vol. 5, supp.3 [1978] for the PAM250 comparison matrix; seeHenikoff et al., Proc. Natl. Acad. Sci USA, 89:10915-10919 [1992] forthe BLOSUM 62 comparison matrix) also may be used by the algorithm. Thepercent identity then is calculated by the algorithm. Homologues willtypically have one or more amino acid substitutions, deletions, and/orinsertions as compared with the comparison NTP protein or NTP peptide,as the case may be.

The term “fusion protein” refers to a protein where one or more NTPpeptides are recombinantly fused or chemically conjugated (includingcovalently and non-covalently) to a protein such as (but not limited to)an antibody or antibody fragment like an Fab fragment or short chain Fv.The term “fusion protein” also refers to multimers (i.e. dimers,trimers, tetramers and higher multimers) of NTP peptides. Such multimerscomprise homomeric multimers comprising one NTP peptide, heteromericmultimers comprising more than one NTP peptide, and heteromericmultimers comprising at least one NTP peptide and at least one otherprotein. Such multimers may be the result of hydrophobic, hyrdrophilic,ionic and/or covalent associations, bonds or links, may be formed bycross-links using linker molecules or may be linked indirectly by, forexample, liposome formation

The term “peptide mimetic” or “mimetic” refers to biologically activecompounds that mimic the biological activity of a peptide or a proteinbut are no longer peptidic in chemical nature, that is, they no longercontain any peptide bonds (that is, amide bonds between amino acids).Here, the term peptide mimetic is used in a broader sense to includemolecules that are no longer completely peptidic in nature, such aspseudo-peptides, semi-peptides and peptoids. Examples of peptidemimetics in this broader sense (where part of a peptide is replaced by astructure lacking peptide bonds) are described below. Whether completelyor partially non-peptide, peptide mimetics according to this inventionprovide a spatial arrangement of reactive chemical moieties that closelyresemble the three-dimensional arrangement of active groups in the NTPpeptide on which the peptide mimetic is based. As a result of thissimilar active-site geometry, the peptide mimetic has effects onbiological systems that are similar to the biological activity of theNTP peptide.

The peptide mimetics of this invention are preferably substantiallysimilar in both three-dimensional shape and biological activity to theNTP peptides described herein. Examples of methods of structurallymodifying a peptide known in the art to create a peptide mimetic includethe inversion of backbone chiral centers leading to D-amino acid residuestructures that may, particularly at the N-terminus, lead to enhancedstability for proteolytical degradation without adversely affectingactivity. An example is given in the paper “Tritriated D-ala¹-Peptide TBinding”, Smith C. S. et al., Drug Development Res., 15, pp. 371-379(1988). A second method is altering cyclic structure for stability, suchas N to C interchain imides and lactames (Ede et al. in Smith and Rivier(Eds.) “Peptides: Chemistry and Biology”, Escom, Leiden (1991), pp.268-270). An example of this is given in conformationally restrictedthymopentin-like compounds, such as those disclosed in U.S. Pat. No.4,457,489 (1985), Goldstein, G. et al., the disclosure of which isincorporated by reference herein in its entirety. A third method is tosubstitute peptide bonds in the NTP peptide by pseudopeptide bonds thatconfer resistance to proteolysis.

A number of pseudopeptide bonds have been described that in general donot affect peptide structure and biological activity. One example ofthis approach is to substitute retro-inverso pseudopeptide bonds(“Biologically active retroinverso analogues of thymopentin”, Sisto A.et al in Rivier, J. E. and Marshall, G. R. (eds) “Peptides, Chemistry,Structure and Biology”, Escom, Leiden (1990), pp. 722-773) and Dalpozzo,et al. (1993), Int. J. Peptide Protein Res., 41:561-566, incorporatedherein by reference). According to this modification, the amino acidsequences of the peptides may be identical to the sequences of an NTPpeptide described above, except that one or more of the peptide bondsare replaced by a retro-inverso pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution willconfer resistance to proteolysis by exopeptidases acting on theN-terminus. Further modifications also can be made by replacing chemicalgroups of the amino acids with other chemical groups of similarstructure. Another suitable pseudopeptide bond that is known to enhancestability to enzymatic cleavage with no or little loss of biologicalactivity is the reduced isostere pseudopeptide bond (Couder, et al.(1993), Int. J. Peptide Protein Res., 41:181-184, incorporated herein byreference in its entirety).

Thus, the amino acid sequences of these peptides may be identical to thesequences of an NTP peptide, except that one or more of the peptidebonds are replaced by an isostere pseudopeptide bond. Preferably themost N-terminal peptide bond is substituted, since such a substitutionwould confer resistance to proteolysis by exopeptidases acting on theN-terminus. The synthesis of peptides with one or more reduced isosterepseudopeptide bonds is known in the art (Couder, et al. (1993), citedabove). Other examples include the introduction of ketomethylene ormethylsulfide bonds to replace peptide bonds.

Peptoid derivatives of NTP peptides represent another class of peptidemimetics that retain the important structural determinants forbiological activity, yet eliminate the peptide bonds, thereby conferringresistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci.USA, 89:9367-9371, incorporated herein by reference in its entirety).Peptoids are oligomers of N-substituted glycines. A number of N-alkylgroups have been described, each corresponding to the side chain of anatural amino acid (Simon, et al. (1992), cited above). Some or all ofthe amino acids of the NTP peptides may be replaced with theN-substituted glycine corresponding to the replaced amino acid.

The term “peptide mimetic” or “mimetic” also includes reverse-D peptidesand enantiomers as defined below.

The term “reverse-D peptide” refers to a biologically active protein orpeptide consisting of D-amino acids arranged in a reverse order ascompared to the L-amino acid sequence of an NTP peptide. Thus, thecarboxy terminal residue of an L-amino acid NTP peptide becomes theamino terminal for the D-amino acid peptide and so forth. For example,the NTP peptide, ETESH (SEQ ID NO: 49), becomesH_(d)S_(d)E_(d)T_(d)E_(d), where E_(d), H_(d), S_(d), and T_(d) are theD-amino acids corresponding to the L-amino acids, E, H, S, and Trespectively.

The term “enantiomer” refers to a biologically active protein or peptidewhere one or more the L-amino acid residues in the amino acid sequenceof an NTP peptide is replaced with the corresponding D-amino acidresidue(s).

A “composition” as used herein, refers broadly to any compositioncontaining a recited peptide or amino acid sequence. The composition maycomprise a dry formulation, an aqueous solution, or a sterilecomposition. Compositions comprising NTP peptides may be employed ashybridization probes. The probes may be stored in freeze-dried form andmay be associated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts, e.g., NaCl, detergents, e.g., sodium dodecyl sulfate(SDS), and other components, e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.

Amino acids and amino acid residues described herein may be referred toaccording to the accepted one or three-letter code provided in the tablebelow. Unless otherwise specified, these amino acids or residues are ofthe naturally occurring L stereoisomer form.

TABLE 1 One-Letter Three-Letter Amino Acid Symbol Symbol Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Methionine M MetPhenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

The present invention is directed to a composition comprising NTPpeptides as defined above in this invention.

A preferred NTP peptide is derived from the amino acid sequence for the122 amino acid sequence of NTP described in FIG. 1 (NTP[122]) or for the112 amino acid sequence of NTP described in FIG. 2 (NTP[112]. However,the use of other NTP peptides based on portions or fragments of othermolecules of the same family as NTP[122] or NTP[112], such as otherneural thread proteins, or such as any of those shown in FIGS. 3-7, andpancreatic thread proteins, also is encompassed by the scope of theinvention. Moreover, the invention includes other proteins that containin whole or part an NTP peptide, whereby the proteins preferably possessthe same, similar, or enhanced bioactivity as the NTP peptide.

Peptide sequences and fragments of AD7c-NTP and other species of NTP andsimilar variants and homologs thereof also are found in a wide varietyof human and non-human proteins (“Related Proteins”). In particular, theAD7c-NTP gene contains Alu-type sequences that are closely similar tothose also found in other genes in the human and other primate genomes.

It is reasonable to assume that some, if not all, of the NTP Peptidesalso will prove to be effective agents for causing cell death becausethey contain peptide sequences identical, homologous or closely similarto peptide sequences found in AD7c-NTP and other species of NTP. Usingthe guidelines provided herein, a person ordinarily skilled in the artcould synthesize specific proteins based on the amino acid sequence forany NTP Peptide found to be an effective agent for causing cell deathand test them for efficacy as agents for causing cell death.

Other peptide sequences derived from a NTP Peptide found to be aneffective agent for causing cell death also may be effective agents forcausing cell death. A person ordinarily skilled in the art can, usingthe guidelines provided herein, synthesize without undue experimentationfragments of an effective NTP Peptide spanning the entire amino acidsequence of that protein in order to identify other effective peptidesequences.

NTP peptides of this invention containing amino acid sequencescorresponding to part of the amino acid sequence for NTP[122] include,but are not limited to, the following:

[SEQ ID NO. 8] NTP[122] peptide #1, NTP[122] p106-122 IDQQVLSRIKLEIKRCLIle-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu- Ile-Lys-Arg-Cys-Leu[SEQ ID NO. 9] NTP[122] peptide #2, NTP[122] p1-15 MMVCWNRFGKWVYFIMet-Met-Val-Cys-Trp-Asn-Arg-Phe-Gly-Lys-Trp-Val- Tyr-Phe-Ile[SEQ ID NO. 10] NTP[122] peptide #3, NTP[122] p16-30 SAIFNFGPRYLYHGVSer-Ala-Ile-Phe-Asn-Phe-Gly-Pro-Arg-Tyr-Leu-Tyr- His-Gly-Val-[SEQ ID NO. 11] NTP[122] peptide #4, NTP[122] p31-45 PFYFLILVRIISFLIPro-Phe-Tyr-Phe-Leu-Ile-Leu-Val-Arg-Ile-Ile-Ser- Phe-Leu-Ile[SEQ ID NO. 12] NTP[122] peptide #5, NTP[122] p46-60 GDMEDVLLNCTLLKRGly-Asp-Met-Glu-Asp-Val-Leu-Leu-Asn-Cys-Thr-Leu- Leu-Lys-Arg[SEQ ID NO. 13] NTP[122] peptide #6, NTP[122] p60-75 SSRFRFWGALVCSMDSer-Ser-Arg-Phe-Arg-Phe-Trp-Gly-Ala-Leu-Val-Cys- Ser-Met-Asp[SEQ ID NO. 14] NTP[122] peptide #7, NTP[122] p76-90 SCRFSRVAVTYRFITSer-Cys-Arg-Phe-Ser-Arg-Val-Ala-Val-Thr-Tyr-Arg- Phe-Ile-Thr[SEQ ID NO. 15] NTP[122] peptide #8, NTP[122] p91-105 LLNIPSPAVWMARNTLeu-Leu-Asn-Ile-Pro-Ser-Pro-Ala-Val-Trp-Met-Ala- Arg-Asn-Thr

NTP peptides of this invention containing amino acid sequencescorresponding to part of the amino acid sequence for NTP[112] include,but are not limited to, the following:

[SEQ ID NO. 16] NTP[112] peptide #1, NTP[112] p1-15 MAQSRLTATSASRVQMet-Ala-Gln-Ser-Arg-Leu-Thr-Ala-Thr-Ser-Ala-Ser- Arg-Val-Gln[SEQ ID NO. 17] NTP[112] peptide #2, NTP[112] p16-30 AILLSQPPKQLGLRAAla-Ile-Leu-Leu-Ser-Gln-Pro-Pro-Lys-Gln-Leu-Gly- Leu-Arg-Ala[SEQ ID NO. 18] NTP[112] peptide #3, NTP[112] p31-45 PANTPLIFVFSLEAGPro-Ala-Asn-Thr-Pro-Leu-Ile-Phe-Val-Phe-Ser-Leu- Glu-Ala-Gly[SEQ ID NO. 19] NTP[112] peptide #4, NTP[112] p46-60 FHHICQAGLKLLTSGPhe-His-His-Ile-Cys-Gln-Ala-Gly-Leu-Lys-Leu-Leu- Thr-Ser-Gly[SEQ ID NO. 20] NTP[112] peptide #5, NTP[112] p61-75 DPPASAFQSAGITGVAsp-Pro-Pro-Ala-Ser-Ala-Phe-Gln-Ser-Ala-Gly-Ile- Thr-Gly-Val[SEQ ID NO. 21] NTP[112] peptide #6, NTP[112] p76-90 SHLTQPANLDKKICSSer-His-Leu-Thr-Gln-Pro-Ala-Asn-Leu-Asp-Lys-Lys- Ile-Cys-Ser[SEQ ID NO. 22] NTP[112] peptide #7, NTP[112] p91-112NGGSCYVAQAGLKLLASCNPSK Asn-Gly-Gly-Ser-Cys-Tyr-Val-Ala-Gln-Ala-Gly-Leu-Lys-Leu-Leu-Ala-Ser-Cys-Asn-Pro-Ser-Lys

NTP peptides of this invention containing amino acid sequencescorresponding to part of the amino acid sequence for the 106 amino acidNTP described in FIG. 3 (NTP[106]) include, but are not limited to, thefollowing:

[SEQ ID NO. 23] NTP[106] peptide #1, NTP[106] p1-15 MWTLKSSLVLLLCLTMet-Trp-Thr-Leu-Lys-Ser-Ser-Leu-Val-Leu-Leu-Leu- Cys-Leu-Thr[SEQ ID NO. 24] NTP[106] peptide #2, NTP[106] p16-30 CSYAFMFSSLRQKTSCys-Ser-Tyr-Ala-Phe-Met-Phe-Ser-Ser-Leu-Arg-Gln- Lys-Thr-Ser[SEQ ID NO. 25] NTP[106] peptide #3, NTP[106] p3145 EPQGKVPCGEHFRIRGlu-Pro-Gln-Gly-Lys-Val-Pro-Cys-Gly-Glu-His-Phe- Arg-Ile-Arg[SEQ ID NO. 26] NTP[106] peptide #4, NTP[106] p46-60 QNLPEHTQGWLGSKWGln-Asn-Leu-Pro-Glu-His-Thr-Gln-Gly-Trp-Leu-Gly- Ser-Lys-Trp[SEQ ID NO. 27] NTP[106] peptide #5, NTP[106] p61-75 LWLLFAVVPFVILKCLeu-Trp-Leu-Leu-Phe-Ala-Val-Val-Pro-Phe-Val-Ile- Leu-Lys-Cys[SEQ ID NO. 28] NTP[106] peptide #6, NTP[106] p76-90 QRDSEKNKVRMAPFFGln-Arg-Asp-Ser-Glu-Lys-Asn-Lys-Val-Arg-Met-Ala- Pro-Phe-Phe[SEQ ID NO. 29] NTP[106] peptide #7, NTP[106] p90-106 LHHIDSISGVSGKRMFLeu-His-His-Ile-Asp-Ser-Ile-Ser-Gly-Val-Ser-Gly- Lys-Arg-Met-Phe

NTP peptides of this invention containing amino acid sequencescorresponding to part of the amino acid sequence for the 98 amino acidNTP described in FIG. 4 (NTP[98]) include, but are not limited to, thefollowing:

[SEQ ID NO. 30] NTP[98] peptide #1, NTP[98] p1-15 EAYYTMLHLPTTNRPGlu-Ala-Tyr-Tyr-Thr-Met-Leu-His-Leu-Pro-Thr-Thr- Asn-Arg-Pro[SEQ ID NO. 31] NTP[98] peptide #2, NTP[98] p16-30 KIAHCILFNQPHSPRLys-Ile-Ala-His-Cys-Ile-Leu-Phe-Asn-Gln-Pro-His- Ser-Pro-Arg-[SEQ ID NO. 32] NTP[98] peptide #3, NTP[98] p31-45 SNSHSHPNPLKLHRRSer-Asn-Ser-His-Ser-His-Pro-Asn-Pro-Leu-Lys-Leu- His-Arg-Arg[SEQ ID NO. 33] NTP[98] peptide #4, NTP[98] p46-60 SHSHNRPRAYILITISer-His-Ser-His-Asn-Arg-Pro-Arg-Ala-Tyr-Ile-Leu- Ile-Thr-Ile[SEQ ID NO. 34] NTP[98] peptide #5, NTP[98] p61-75 LPSKLKLRTHSQSHHLeu-Pro-Ser-Lys-Leu-Lys-Leu-Arg-Thr-His-Ser-Gln- Ser-His-His[SEQ ID NO. 35] NTP[98] peptide #6, NTP[98] p76-98NPLSRTSNSTPTNSFLMTSSKPR Asn-Pro-Leu-Ser-Arg-Thr-Ser-Asn-Ser-Thr-Pro-Thr-Asn-Ser-Phe-Leu-Met-Thr-Ser-Ser-Lys-Pro-Arg

NTP peptides of this invention containing amino acid sequencescorresponding to part of the amino acid sequence for the 75 amino acidNTP described in FIG. 5 (NTP[75]) include, but are not limited to, thefollowing:

[SEQ ID NO. 36] NTP[75] peptide #1, NTP[75] p1-15 SSSLGLPKCWDYRHESer-Ser-Ser-Leu-Gly-Leu-Pro-Lys-Cys-Trp-Asp-Tyr- Arg-His-Glu[SEQ ID NO. 37] NTP[75] peptide #2, NTP[75] p16-30 LLSLALMINFRVMACLeu-Leu-Ser-Leu-Ala-Leu-Met-Ile-Asn-Phe-Arg-Val- Met-Ala-Cys[SEQ ID NO. 38] NTP[75] peptide #3, NTP[75] p31-45 TFKQHIELRQKISIVThr-Phe-Lys-Gln-His-Ile-Glu-Leu-Arg-Gln-Lys-Ile- Ser-Ile-Val[SEQ ID NO. 39] NTP[75] peptide #4, NTP[75] p46-60 PRKLCCMGPVCPVKIPro-Arg-Lys-Leu-Cys-Cys-Met-Gly-Pro-Val-Cys-Pro- Val-Lys-Ile[SEQ ID NO. 40] NTP[75] peptide #5, NTP[75] p61-75 ALLTINGHCTWLPASAla-Leu-Leu-Thr-Ile-Asn-Gly-His-Cys-Thr-Trp-Leu- Pro-Ala-Ser

NTP peptides of this invention containing amino acid sequencescorresponding to part of the amino acid sequence for the 68 amino acidNTP described in FIG. 6 (NTP[68]) include, but are not limited to, thefollowing:

[SEQ ID NO. 41] NTP[68] peptide #1, NTP[68] p1-15 MFVFCLILNREKIKGMet-Phe-Val-Phe-Cys-Leu-Ile-Leu-Asn-Arg-Glu-Lys- Ile-Lys-Gly[SEQ ID NO. 42] NTP[68] peptide #2, NTP[68] p16-30 GNSSFFLLSFFFSFQGly-Asn-Ser-Ser-Phe-Phe-Leu-Leu-Ser-Phe-Phe-Phe- Ser-Phe-Gln[SEQ ID NO. 43] NTP[68] peptide #3, NTP[68] p31-45 NCCQCFQCRTTEGYAAsn-Cys-Cys-Gln-Cys-Phe-Gln-Cys-Arg-Thr-Thr-Glu- Gly-Tyr-Ala[SEQ ID NO. 44] NTP[68] peptide #4, NTP[68] p46-68VECFYCLVDKAAFECWWFYSFDT Val-Glu-Cys-Phe-Tyr-Cys-Leu-Val-Asp-Lys-Ala-Ala-Phe-Glu-Cys-Trp-Trp-Phe-Tyr-Ser-Phe-Asp-Thr

NTP peptides of this invention containing amino acid sequencescorresponding to part of the amino acid sequence for the 61 amino acidNTP described in FIG. 7 (NTP[61]) include, but are not limited to, thefollowing:

[SEQ ID NO. 45] NTP[61] peptide #1, NTP[61] p1-15 MEPHTVAQAGVPQHDMet-Glu-Pro-His-Thr-Val-Ala-Gln-Ala-Gly-Val-Pro- Gln-His-Asp[SEQ ID NO. 46] NTP[61] peptide #2, NTP[61] p16-30 LGSLQSLLPRFKRFSLeu-Gly-Ser-Leu-Gln-Ser-Leu-Leu-Pro-Arg-Phe-Lys- Arg-Phe-Ser[SEQ ID NO. 47] NTP[61] peptide #3, NTP[61] p31-45 CLILPKIWDYRNMNTCys-Leu-Ile-Leu-Pro-Lys-Ile-Trp-Asp-Tyr-Arg-Asn- Met-Asn-Thr[SEQ ID NO. 48] NTP[61] peptide #4, NTP[61] p46-61 ALIKRNRYTPETGRKSAla-Leu-Ile-Lys-Arg-Asn-Arg-Tyr-Thr-Pro-Glu-Thr- Gly-Arg-Lys-Ser

It will be apparent to one of skill in the art that other smallerfragments of the above NTP peptides may be selected such that thesepeptides will possess the same or similar biological activity. Otherfragments of NTP may be selected by one skilled in the art such thatthese peptides will possess the same or similar biological activity. TheNTP peptides of the invention encompass these other fragments. Ingeneral, the peptides of this invention have at least 6 amino acids,preferably at least 5 amino acids, and more preferably at least 4 aminoacids.

The invention also encompasses peptides comprising two or more NTPpeptides joined together, even if the sequences of the two NTP peptidesare not contiguous in the sequence of the specie(s) of NTP from whichthe NTP peptides were derived. To the extent that an NTP peptide has thedesired biological activity, it follows that two such NTP peptides wouldalso possess the desired biological activity, even if these segmentswere not contiguous within the sequence of amino acids of the specie(s)of NTP from which the NTP peptides were derived.

NTP peptides and fragments, variants, derivatives, homologues, fusionproteins and mimetics thereof encompassed by this invention can beprepared using methods known to those of skill in the art, such asrecombinant DNA technology, protein synthesis and isolation of naturallyoccurring NTP peptides, NTP proteins, AD7c-NTP protein and fragments,variants, derivatives and homologues thereof.

NTP peptides and fragments, variants, derivatives, homologues, fusionproteins and mimetics thereof can be prepared from other NTP peptides,NTP proteins, AD7c-NTP proteins and fragments, variants, derivatives andhomologues thereof using methods known to those having skill in the art.Such methods include (but are not limited to) the use of proteases tocleave the NTP peptide, NTP protein or AD7c-NTP protein into the desiredNTP peptide.

An NTP peptide or an NTP protein can be prepared using well knownrecombinant DNA technology methods such as those set forth in Sambrooket al. Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1989] and/or Ausubel et al.,eds., Current Protocols in Molecular Biology, Green Publishers Inc. andWiley and Sons, N.Y. [1994].

A gene or cDNA encoding an NTP peptide or an NTP protein may be obtainedfor example by screening a genomic or cDNA library, or by PCRamplification. Probes or primers useful for screening the library can begenerated based on sequence information for other known genes or genefragments from the same or a related family of genes, such as, forexample, conserved motifs found in other NTP peptides or NTP proteins.In addition, where a gene encoding an NTP peptide or NTP protein hasbeen identified from one species, all or a portion of that gene may beused as a probe to identify homologous genes from other species. Theprobes or primers may be used to screen cDNA libraries from varioustissue sources believed to express an NTP peptide or NTP protein gene.Typically, conditions of high stringency will be employed for screeningto minimize the number of false positives obtained from the screen.

Another means to prepare a gene encoding an NTP peptide or NTP proteinis to employ chemical synthesis using methods well known to the skilledartisan, such as those described by Engels et al., Angew. Chem. Intl.Ed., 28:716-734 [1989]. These methods include, inter alia, thephosphotriester, phosphoramidite, and H-phosphonate methods for nucleicacid synthesis. A preferred method for such chemical synthesis ispolymer-supported synthesis using standard phosphoramidite chemistry.Typically, the DNA encoding an NTP peptide or NTP protein will beseveral hundred nucleotides in length. Nucleic acids larger than about100 nucleotides can be synthesized as several fragments using thesemethods. The fragments then can be ligated together to form the fulllength NTP peptide or NTP protein. Usually, the DNA fragment encodingthe amino terminus of the protein will have an ATG, which encodes amethionine residue. This methionine may or may not be present on themature form of the NTP protein or NTP peptide, depending on whether theprotein produced in the host cell is designed to be secreted from thatcell.

The gene, cDNA, or fragment thereof encoding the NTP protein or NTPpeptide can be inserted into an appropriate expression or amplificationvector using standard ligation techniques. The vector is typicallyselected to be functional in the particular host cell employed (i.e.,the vector is compatible with the host cell machinery such thatamplification of the gene and/or expression of the gene can occur). Thegene, cDNA or fragment thereof encoding the NTP protein or NTP peptidemay be amplified/expressed in prokaryotic, yeast, insect (baculovirussystems) and/or eukaryotic host cells. Selection of the host cell willdepend in part on whether the NTP protein or NTP peptide is to beglycosylated and/or phosphorylated. If so, yeast, insect, or mammalianhost cells are preferable.

Typically, the vectors used in any of the host cells will contain atleast a 5′ flanking sequence (also referred to as a promoter) and otherregulatory elements as well, such as an enhancer(s), an origin ofreplication element, a transcriptional termination element, a completeintron sequence containing a donor and acceptor splice site, a signalpeptide sequence, a ribosome binding site element, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these elements is discussed below. Optionally, the vector may containa tag sequence, i.e., an oligonucleotide molecule located at the 5′ or3′ end of the NTP protein or NTP peptide coding sequence; theoligonucleotide molecule encodes polyHis (such as hexaHis), or other tagsuch as FLAG, HA (hemaglutinin Influenza virus) or myc for whichcommercially available antibodies exist. This tag is typically fused tothe polypeptide upon expression of the polypeptide, and can serve asmeans for affinity purification of the NTP protein or NTP peptide fromthe host cell. Affinity purification can be accomplished, for example,by column chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified NTP protein or NTP peptide by various means such as usingcertain peptidases.

The human immunoglobulin hinge and Fc region could be fused at eitherthe N-terminus or C-terminus of the NTP protein or NTP peptide by oneskilled in the art. The subsequent Fc-fusion protein could be purifiedby use of a Protein A affinity column. Fc is known to exhibit a longpharmacokinetic half-life in vivo and proteins fused to Fc have beenfound to exhibit a substantially greater half-life in vivo than theunfused counterpart. Also, fusion to the Fc region allows fordimerization/multimerization of the molecule that may be useful for thebioactivity of some molecules.

The 5′ flanking sequence may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof 5′ flanking sequences from more than one source), synthetic, or itmay be the native NTP protein or NTP peptide gene 5′ flanking sequence.As such, the source of the 5′ flanking sequence may be any unicellularprokaryotic or eukaryotic organism, any vertebrate or invertebrateorganism, or any plant, provided that the 5′ flanking sequence isfunctional in, and can be activated by, the host cell machinery.

The 5′ flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically, 5′flanking sequences useful herein other than the NTP protein or NTPpeptide gene flanking sequence will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of the 5′flanking sequence may be known. Here, the 5′ flanking sequence may besynthesized using the methods described above for nucleic acid synthesisor cloning.

Where all or only a portion of the 5′ flanking sequence is known, it maybe obtained using PCR and/or by screening a genomic library withsuitable oligonucleotide and/or 5′ flanking sequence fragments from thesame or another species.

Where the 5′ flanking sequence is not known, a fragment of DNAcontaining a 5′ flanking sequence may be isolated from a larger piece ofDNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion using one or more carefully selected enzymes to isolate theproper DNA fragment. After digestion, the desired fragment may beisolated by agarose gel purification, Qiagen® column or other methodsknown to the skilled artisan. Selection of suitable enzymes toaccomplish this purpose will be readily apparent to one of ordinaryskill in the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe NTP protein or NTP peptide. If the vector of choice does not containan origin of replication site, one may be chemically synthesized basedon a known sequence, and ligated into the vector. The transcriptiontermination element is typically located 3′ of the end of the NTPprotein or NTP peptide coding sequence and serves to terminatetranscription of the NTP protein or NTP peptide. Usually, thetranscription termination element in prokaryotic cells is a G-C richfragment followed by a poly T sequence. While the element may be clonedfrom a library or purchased commercially as part of a vector, it canalso be readily synthesized using methods for nucleic acid synthesissuch as those described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (prokaryotes) or the Kozak sequence (eukaryotes), is usuallynecessary for translation initiation of mRNA. The element is typicallylocated 3′ to the promoter and 5′ to the coding sequence of the NTPprotein or NTP peptide to be synthesized. The Shine-Dalgarno sequence isvaried but is typically a polypurine (i.e., having a high A-G content).Many Shine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In those cases where it is desirable for NTP protein or NTP peptide tobe secreted from the host cell, a signal sequence may be used to directthe NTP protein or NTP peptide out of the host cell where it issynthesized, and the carboxy-terminal part of the protein may be deletedin order to prevent membrane anchoring. Typically, the signal sequenceis positioned in the coding region of the NTP protein/NTP peptide geneor cDNA, or directly at the 5′ end of the NTP protein/NTP peptide genecoding region. Many signal sequences have been identified, and any ofthem that are functional in the selected host cell may be used inconjunction with the NTP protein/NTP peptide gene or cDNA. Therefore,the signal sequence may be homologous or heterologous to the NTPprotein/NTP peptide gene or cDNA, and may be homologous or heterologousto the NTP protein/NTP peptide gene or cDNA. Additionally, the signalsequence may be chemically synthesized using methods set forth above. Inmost cases, secretion of the polypeptide from the host cell via thepresence of a signal peptide will result in the removal of the aminoterminal methionine from the polypeptide.

In many cases, transcription of the NTP protein/NTP peptide gene or cDNAis increased by the presence of one or more introns in the vector; thisis particularly true where the NTP protein or NTP peptide is produced ineukaryotic host cells, especially mammalian host cells. The introns usedmay be naturally occurring within the NTP protein/NTP peptide gene,especially where the gene used is a full length genomic sequence or afragment thereof. Where the intron is not naturally occurring within thegene (as for most cDNAs), the intron(s) may be obtained from anothersource. The position of the intron with respect to the flanking sequenceand the NTP protein/NTP peptide gene generally is important, as theintron must be transcribed to be effective. As such, where the NTPprotein/NTP peptide gene inserted into the expression vector is a cDNAmolecule, the preferred position for the intron is 3′ to thetranscription start site, and 5′ to the polyA transcription terminationsequence. Preferably for NTP protein/NTP peptide cDNA, the intron orintrons will be located on one side or the other (i.e., 5′ or 3′) of thecDNA such that it does not interrupt this coding sequence. Any intronfrom any source, including any viral, prokaryotic and eukaryotic (plantor animal) organisms, may be used to practice this invention, providedthat it is compatible with the host cell(s) into which it is inserted.Also included herein are synthetic introns. Optionally, more than oneintron may be used in the vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan and are comparable to the methodsset forth above (i.e., synthesis of the DNA, library screening, and thelike).

The final vectors used to practice this invention may be constructedfrom starting vectors such as a commercially available vector. Suchvectors may or may not contain some of the elements to be included inthe completed vector. If none of the desired elements are present in thestarting vector, each element may be individually ligated into thevector by cutting the vector with the appropriate restrictionendonuclease(s) such that the ends of the element to be ligated in andthe ends of the vector are compatible for ligation. In some cases, itmay be necessary to blunt the ends to be ligated together in order toobtain a satisfactory ligation. Blunting is accomplished by firstfilling in “sticky ends” using Klenow DNA polymerase or T4 DNApolymerase in the presence of all four nucleotides. This procedure iswell known in the art and is described for example in Sambrook et al.,supra. Alternatively, two or more of the elements to be inserted intothe vector may first be ligated together (if they are to be positionedadjacent to each other) and then ligated into the vector.

An additional method for constructing the vector is to conduct allligations of the various elements simultaneously in one reactionmixture. Here, many nonsense or nonfunctional vectors will be generateddue to improper ligation or insertion of the elements, however thefunctional vector may be identified and selected by restrictionendonuclease digestion.

Preferred vectors for practicing this invention are those that arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, San Diego, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15b (Novagen, Madison, Wis.), PGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII; Invitrogen), and pFastBacDual (Gibco/BRL, Grand Island,N.Y.).

After the vector has been constructed and a nucleic acid moleculeencoding full length or truncated NTP protein or NTP peptide has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or polypeptideexpression. Host cells may be prokaryotic host cells (such as E. coli)or eukaryotic host cells (such as a yeast cell, an insect cell, or avertebrate cell). The host cell, when cultured under appropriateconditions, can synthesize NTP protein or NTP peptide which cansubsequently be collected from the culture medium (if the host cellsecretes it into the medium) or directly from the host cell producing it(if it is not secreted).

After collection, the NTP protein or NTP peptide can be purified usingmethods such as molecular sieve chromatography, affinity chromatography,and the like. Selection of the host cell for NTP protein or NTP peptideproduction will depend in part on whether the NTP protein or NTP peptideis to be glycosylated or phosphorylated (in which case eukaryotic hostcells are preferred), and the manner in which the host cell is able tofold the protein into its native tertiary structure (e.g., properorientation of disulfide bridges, etc.) such that biologically activeprotein is prepared by the NTP protein or NTP peptide that hasbiological activity, the NTP protein or NTP peptide may be folded aftersynthesis using appropriate chemical conditions as discussed below.Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO), human embryonic kidney (HEK) 293, 293T cells,or 3T3 cells. The selection of suitable mammalian host cells and methodsfor transformation, culture, amplification, screening and productproduction and purification are known in the art. Other suitablemammalian cell lines, are the monkey COS-1 and COS-7 cell lines, and theCV-1 cell line. Further exemplary mammalian host cells include primatecell lines and rodent cell lines, including transformed cell lines.Normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, as well as primary explants, are also suitable.Candidate cells may be genotypically deficient in the selection gene, ormay contain a dominantly acting selection gene. Other suitable mammaliancell lines include but are not limited to, mouse neuroblastoma N2Acells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c orNIH mice, BHK or HaK hamster cell lines.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5.alpha., DH10, and MC1061) are well-known as host cells in thefield of biotechnology. Various strains of B. subtilis, Pseudomonasspp., other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method. Many strains of yeast cells known to thoseskilled in the art also are available as host cells for expression ofthe polypeptides of the present invention.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al. (Biotechniques, 14:810-817 [1993]), Lucklow (Curr. Opin.Biotechnol., 4:564-572 [1993]) and Lucklow of al. (J. Virol.,67:4566-4579 [1993]). Preferred insect cells are Sf-9 and Hi5(Invitrogen, Carlsbad, Calif.).

Insertion (also referred to as transformation or transfection) of thevector into the selected host cell may be accomplished using suchmethods as calcium chloride, electroporation, microinjection,lipofection, or the DEAE-dextran method. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., supra.

The host cells containing the vector (i.e., transformed or transfected)may be cultured using standard media well known to the skilled artisan.The media will usually contain all nutrients necessary for the growthand survival of the cells. Suitable media for culturing E. coli cellsare for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitablemedia for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all ofwhich may be supplemented with serum and/or growth factors as requiredby the particular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary. Typically, anantibiotic or other compound useful for selective growth of thetransformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of NTP protein or NTP peptide produced in the host cell canbe evaluated using standard methods known in the art. Such methodsinclude, without limitation, Western blot analysis, SDS-polyacrylamidegel electrophoresis, non-denaturing gel electrophoresis, HPLCseparation, mass spectroscopy, immunoprecipitation, and/or activityassays such as DNA binding gel shift assays.

If the NTP protein or NTP peptide has been designed to be secreted fromthe host cells, the majority of the NTP protein or NTP peptide may befound in the cell culture medium. Proteins prepared in this way willtypically not possess an amino terminal methionine, as it is removedduring secretion from the cell. If however, the NTP protein or NTPpeptide is not secreted from the host cells, it will be present in thecytoplasm and/or the nucleus (for eukaryotic host cells) or in thecytosol (for gram negative bacteria host cells) and may have an aminoterminal methionine.

For NTP protein or NTP peptide situated in the host cell cytoplasmand/or nucleus, the host cells are typically first disruptedmechanically or with detergent to release the intra-cellular contentsinto a buffered solution. NTP protein or NTP peptide can then beisolated from this solution.

Purification of NTP protein or NTP peptide from solution can beaccomplished using a variety of techniques. If the protein has beensynthesized such that it contains a tag such as hexaHistidine (e.g. NTPpeptide/hexaHis) or other small peptide such as FLAG (Sigma-Aldritch,St. Louis, Mich.) or calmodulin-binding peptide (Stratagene, La Jolla,Calif.) at either its carboxyl or amino terminus, it may essentially bepurified in a one-step process by passing the solution through anaffinity column where the column matrix has a high affinity for the tagor for the protein directly (i.e., a monoclonal antibody specificallyrecognizing the NTP peptide). For example, polyhistidine binds withgreat affinity and specificity to nickel, zinc and cobalt; thusimmobilized metal ion affinity chromatography which employs anickel-based affinity resin (as used in Qiagen's QIAexpress system orInvitrogen's Xpress System) or a cobalt-based affinity resin (as used inBD Biosciences-CLONTECH's Talon system) can be used for purification ofNTP peptide/polyHis. (See, for example, Ausubel et al., eds., CurrentProtocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, NewYork [1993]).

Where the NTP protein or NTP peptide is prepared without a tag attached,and no antibodies are available, other well known procedures forpurification can be used. Such procedures include, without limitation,ion exchange chromatography, hydroxyapatite chromatography, hydrophobicinteraction chromatography, molecular sieve chromatography, HPLC, nativegel electrophoresis in combination with gel elution, and preparativeisoelectric focusing (Isoprime machine/technique, Hoefer Scientific). Insome cases, two or more of these techniques may be combined to achieveincreased purity.

If it is anticipated that the NTP protein or NTP peptide will be foundprimarily intracellularly, the intracellular material (includinginclusion bodies for gram-negative bacteria) can be extracted from thehost cell using any standard technique known to the skilled artisan. Forexample, the host cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation. If the NTP protein or NTP peptide has formedinclusion bodies in the cytosol, the inclusion bodies can often bind tothe inner and/or outer cellular membranes and thus will be foundprimarily in the pellet material after centrifugation. The pelletmaterial then can be treated at pH extremes or with chaotropic agentsuch as a detergent, guanidine, guanidine derivatives, urea, or ureaderivatives in the presence of a reducing agent such as dithiothreitolat alkaline pH or tris carboxyethyl phosphine at acid pH to release,break apart, and solubilize the inclusion bodies. The NTP protein or NTPpeptide in its now soluble form can then be analyzed using gelelectrophoresis, immunoprecipitation or the like. If it is desired toisolate the NTP protein or NTP peptide, isolation may be accomplishedusing standard methods such as those set forth below and in Marston etal. Meth. Enz., 182:264-275 (1990).

In some cases, the NTP protein or NTP peptide may not be biologicallyactive upon isolation. Various methods for refolding or converting thepolypeptide to its tertiary structure and generating disulfide linkages,can be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization but usually at a lower concentration and is notnecessarily the same chaotrope as used for the solubilization. In mostcases the refolding/oxidation solution will also contain a reducingagent or the reducing agent plus its, oxidized form in a specific ratioto generate a particular redox potential allowing for disulfideshuffling to occur in the formation of the protein's cysteine bridge(s).Some of the commonly used redox couples include cysteine/cystamine,glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, 2-mercaptoethanol(bME)/dithio-b(ME).In many instances a cosolvent is necessary to increase the efficiency ofthe refolding and the more common reagents used for this purpose includeglycerol, polyethylene glycol of various molecular weights, andarginine.

If NTP protein or NTP peptide inclusion bodies are not formed to asignificant degree in the host cell, the NTP protein or NTP peptide willbe found primarily in the supernatant after centrifugation of the cellhomogenate, and the NTP protein or NTP peptide can be isolated from thesupernatant using methods such as those set forth below.

In those situations where it is preferable to partially or completelyisolate the NTP protein or NTP peptide, purification can be accomplishedusing standard methods well known to the skilled artisan. Such methodsinclude, without limitation, separation by electrophoresis followed byelectroelution, various types of chromatography (immunoaffinity,molecular sieve, and/or ion exchange), and/or high pressure liquidchromatography. In some cases, it may be preferable to use more than oneof these methods for complete purification.

In addition to preparing and purifying NTP proteins or NTP peptidesusing recombinant DNA techniques, the NTP proteins or NTP peptides andtheir fragments, variants, homologues, fusion proteins, peptidemimetics, and derivatives may be prepared by chemical synthesis methods(such as solid phase peptide synthesis) using techniques known in theart such as those set forth by Merrifield et al., J. Am. Chem. Soc.,85:2149 [1963], Houghten et al. Proc Natl Acad. Sci. USA, 82:5132[1985], and Stewart and Young, Solid Phase Peptide Synthesis, PierceChemical Co., Rockford, Ill. [1984]. Such polypeptides may besynthesized with or without a methionine on the amino terminus.Chemically synthesized NTP proteins or NTP peptides may be oxidizedusing methods set forth in these references to form disulfide bridges.The NTP proteins or NTP peptides are expected to have biologicalactivity comparable to NTP proteins or NTP peptides producedrecombinantly or purified from natural sources, and thus may be usedinterchangeably with recombinant or natural NTP protein or NTP peptide.

Chemically modified NTP peptide compositions in which the NTP peptide islinked to a polymer are included within the scope of the presentinvention. The polymer selected is typically water soluble so that theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. The polymer selectedis usually modified to have a single reactive group, such as an activeester for acylation or an aldehyde for alkylation, so that the degree ofpolymerization may be controlled as provided for in the present methods.The polymer may be of any molecular weight, and may be branched orunbranched. Included within the scope of NTP peptide polymers is amixture of polymers.

In some cases, it may be desirable to prepare nucleic acid and/or aminoacid variants of the naturally occurring NTP proteins or NTP peptides.Nucleic acid variants may be produced using site directed mutagenesis,PCR amplification, or other appropriate methods, where the primer(s)have the desired point mutations (see Sambrook et al., supra, andAusubel et al., supra, for descriptions of mutagenesis techniques).Chemical synthesis using methods described by Engels et al., supra, mayalso be used to prepare such variants. Other methods known to theskilled artisan may be used as well.

Preferred nucleic acid variants are those containing nucleotidesubstitutions accounting for codon preference in the host cell that isto be used to produce the NTP protein or NTP peptide. Such codonoptimization can be determined via computer algorithers whichincorporate codon frequency tables such as Ecohigh. Cod for codonpreference of highly expressed bacterial genes as provided by theUniversity of Wisconsin Package Version 9.0, Genetics Computer Group,Madison, Wis. Other useful codon frequency tables includeCelegans_high.cod, Celegans_low.cod, Drosophila_high.cod,Human_high.cod, Maize_high.cod, and Yeast_high.cod. Other preferredvariants are those encoding conservative amino acid changes as describedabove (e.g., wherein the charge or polarity of the naturally occurringamino acid side chain is not altered substantially by substitution witha different amino acid) as compared to wild type, and/or those designedto either generate a novel glycosylation and/or phosphorylation site(s),or those designed to delete an existing glycosylation and/orphosphorylation site(s).

NTP proteins, NTP peptides, and fragments, homologs, variants, fusionproteins, peptide mimetics, derivatives and salts thereof also can bemade using conventional peptide synthesis techniques known to theskilled artisan. These techniques include chemical coupling methods (cf.Wunsch, E: “Methoden der organischen Chemie”, Volume 15, Band 1+2,Synthese von Peptiden, thime Verlag, Stuttgart (1974), and Barrany, G.;Marrifield, R. B.: “The Peptides,” eds. E. Gross, J. Meienhofer, Volume2, Chapter 1, pp. 1-284, Academic Press (1980)), enzymatic couplingmethods (cf. Widmer, F. Johansen, J. T., Carlsberg Res. Commun., Vol.44, pp. 37-46 (1979); Kullmann, W.: “Enzymatic Peptide Synthesis”, CRCPress Inc. Boca Raton, Fla. (1987); and Widmer, F., Johansen, J. T. in“Synthetic Peptides in Biology and Medicines,” eds. Alitalo, K.,Partanen, P., Vatieri, A., pp. 79-86, Elsevier, Amsterdam (1985)), or acombination of chemical and enzymatic methods if this is advantageousfor the process design and economy. Using the guidelines providedherein, those skilled in the art are capable of varying the peptidesequence of the NTP peptide to make a homologue having the same orsimilar biological activity (bioactivity) as the original or native NTPprotein or NTP peptide.

Advantages exist for using a mimetic of a given NTP peptide rather thanthe peptide itself. In general, peptide mimetics are more bioavailable,have a longer duration of action and can be cheaper to produce than thenative proteins and peptides.

Thus the NTP peptides described above have utility in the development ofsuch small chemical compounds with similar biological activities andtherefore with similar therapeutic utilities. Peptide mimetics of NTPpeptides can be developed using combinatorial chemistry techniques andother techniques known in the art (see e.g. Proceedings of the 20thEuropean Peptide Symposium, ed. G. Jung, E. Bayer, pp. 289-336, andreferences therein).

Examples of methods known in the art for structurally modifying apeptide to create a peptide mimetic include the inversion of backbonechiral centers leading to D-amino acid residue structures that may,particularly at the N-terminus, lead to enhanced stability forproteolytical degradation without adversely affecting activity. Anexample is provided in the paper “Tritriated D-ala¹-Peptide T Binding”,Smith C. S. et al., Drug Development Res. 15, pp. 371-379 (1988).

A second method is altering cyclic structure for stability, such as N toC interchain imides and lactames (Ede et al. in Smith and Rivier (Eds.)“Peptides: Chemistry and Biology”, Escom, Leiden (1991), pp. 268-270).An example of this is given in conformationally restrictedthymopentin-like compounds, such as those disclosed in U.S. Pat. No.4,457,489 (1985), Goldstein, G. et al., the disclosure of which isincorporated by reference herein in its entirety.

A third method is to substitute peptide bonds in the NTP peptide bypseudopeptide bonds that confer resistance to proteolysis. A number ofpseudopeptide bonds have been described that in general do not affectpeptide structure and biological activity. One example of this approachis to substitute retro-inverso pseudopeptide bonds (“Biologically activeretroinverso analogues of thymopentin”, Sisto A. et al in Rivier, J. E.and Marshall, G. R. (eds) “Peptides, Chemistry, Structure and Biology”,Escom, Leiden (1990), pp. 722-773) and Dalpozzo, et al. (1993), Int. J.Peptide Protein Res., 41:561-566, incorporated herein by reference).According to this modification, the amino acid sequences of the peptidesmay be identical to the sequences of the NTP peptides described above,except that one or more of the peptide bonds are replaced by aretro-inverso pseudopeptide bond. Preferably the most N-terminal peptidebond is substituted, since such a substitution will confer resistance toproteolysis by exopeptidases acting on the N-terminus.

The synthesis of peptides with one or more reduced retro-inversopseudopeptide bonds is known in the art (Sisto (1990) and Dalpozzo, etal. (1993), cited above). Thus, peptide bonds can be replaced bynon-peptide bonds that allow the peptide mimetic to adopt a similarstructure, and therefore biological activity, to the original peptide.Further modifications also can be made by replacing chemical groups ofthe amino acids with other chemical groups of similar structure. Anothersuitable pseudopeptide bond that is known to enhance stability toenzymatic cleavage with no or little loss of biological activity is thereduced isostere pseudopeptide bond is a (Couder, et al. (1993), Int. J.Peptide Protein Res., 41:181-184, incorporated herein by reference inits entirety). Thus, the amino acid sequences of these peptides may beidentical to the sequences of an NTP peptide, except that one or more ofthe peptide bonds are replaced by an isostere pseudopeptide bond.Preferably the most N-terminal peptide bond is substituted, since such asubstitution would confer resistance to proteolysis by exopeptidasesacting on the N-terminus. The synthesis of peptides with one or morereduced isostere pseudopeptide bonds is known in the art (Couder, et al.(1993), cited above). Other examples include the introduction ofketomethylene or methylsulfide bonds to replace peptide bonds.

Peptoid derivatives of NTP peptides represent another class of peptidemimetics that retain the important structural determinants forbiological activity, yet eliminate the peptide bonds, thereby conferringresistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci.USA, 89:9367-9371 and incorporated herein by reference in its entirety).Peptoids are oligomers of N-substituted glycines. A number of N-alkylgroups have been described, each corresponding to the side chain of anatural amino acid (Simon, et al. (1992), cited above and incorporatedherein by reference in its entirety). Some or all of the amino acids ofthe NTP peptide are replaced with the N-substituted glycinecorresponding to the replaced amino acid.

The development of peptide mimetics can be aided by determining thetertiary structure of the original NTP peptide by NMR spectroscopy,crystallography and/or computer-aided molecular modeling. Thesetechniques aid in the development of novel compositions of higherpotency and/or greater bioavailability and/or greater stability than theoriginal peptide (Dean (1994), BioEssays, 16: 683-687; Cohen andShatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley and Rich (1993),Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15:124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993),Sci. Ani., 269: 92-98, all incorporated herein by reference in theirentirety).

Once a potential peptide mimetic compound is identified, it may besynthesized and assayed using the methods outlined in the examples belowto assess its activity. The peptide mimetic compounds obtained by theabove methods, having the biological activity of the NTP peptides andsimilar three-dimensional structure, are encompassed by this invention.It will be readily apparent to one skilled in the art that a peptidemimetic can be generated from any of the NTP peptides bearing one ormore of the modifications described above. It will furthermore beapparent that the peptide mimetics of this invention can be further usedfor the development of even more potent non-peptidic compounds, inaddition to their utility as therapeutic compounds.

A number of organizations exist today that are capable of synthesizingthe NTP peptides described herein. For example, given the sequence of anNTP peptide, the organization can synthesize the peptide and forward thesynthesized peptide with accompanying documentation and proof of theidentity of the peptide.

This invention also encompasses the use of NTP peptides and theircorresponding nucleic acid molecules for assays to test, eitherqualitatively or quantitatively, for the presence of NTP peptides, NTPproteins, AD7c-NTP, NTP peptide DNA, NTP protein DNA, AD7c-NTP DNA orcorresponding RNA in mammalian tissue or bodily fluid samples. NTPpeptides and their corresponding nucleic acid molecules may have use inthe preparation in such assays, whether or not the NTP peptide or theencoded NTP peptide DNA show biological activity. NTP peptide nucleicacid sequences may be a useful source of hybridization probes to test,either qualitatively or quantitatively, for the presence of NTP peptideDNA, NTP protein DNA, AD7c-NTP DNA or corresponding RNA in mammaliantissue or bodily fluid samples. NTP peptide which is not in itselfbiologically active may be useful for preparing antibodies thatrecognize and/or bind to NTP peptides, NTP proteins or AD7c-N11 protein.Such antibodies may be prepared using standard methods. Thus, antibodiesthat react with or bind to the NTP peptides, as well as short chainantibody fragments and other reactive fragments of such antibodies, alsoare contemplated as within the scope of the present invention. Theantibodies may be polyclonal, monoclonal, recombinant, chimeric,single-chain and/or bispecific. Typically, the antibody or fragmentthereof will either be of human origin, or will be humanized, i.e.,prepared so as to prevent or minimize an immune reaction to the antibodywhen administered to a patient. Preferred antibodies are humanantibodies, either polyclonal or monoclonal. The antibody fragment maybe any fragment that is reactive with NTP peptides of the presentinvention, such as, Fab, Fab′, etc. Also provided by this invention arethe hybridomas generated by presenting any NTP peptide as an antigen toa selected mammal, followed by fusing cells (e.g., spleen cells) of themammal with certain cancer cells to create immortalized cell lines byknown techniques. The methods employed to generate such cell lines andantibodies directed against all or portions of an NTP peptide are alsoencompassed by this invention.

The antibodies may further be used for in vivo and in vitro diagnosticor research purposes, such as in labeled form to detect the presence ofNTP peptide, NTP protein or AD7c-NTP in a body fluid or cell sample.

This invention also encompasses the use of one or more NTP peptides ascalibration standards in assays that test, either qualitatively orquantitatively, for the presence of NTP peptides, NTP proteins,AD7c-NTP, NTP peptide DNA, NTP protein DNA, AD7c-NTP DNA orcorresponding RNA in mammalian tissue or bodily fluid samples.

The present invention is directed to methods of treating conditionsrequiring removal of cells, such as benign and malignant tumors,glandular (e.g. prostate) hyperplasia, unwanted facial hair, warts, andunwanted fatty tissue, or the inhibition or prevention of unwantedcellular proliferation, such as stenosis of a stent. Such a methodcomprises administering to a mammal in need, or coating a device such asa stent with, a therapeutically effective amount of NTP peptide.

The condition can be, for example, tumors of lung, breast, stomach,pancreas, prostate, bladder, bone, ovary, skin, kidney, sinus, colon,intestine, stomach, rectum, esophagus, blood, brain and its coverings,spinal cord and its coverings, muscle, connective tissue, adrenal,parathyroid, thyroid, uterus, testis, pituitary, reproductive organs,liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, lymph nodesand lymphoid system, and other organs.

As used herein, the term “malignant tumor” is intended to encompass allforms of human carcinomas, sarcomas and melanomas which occur in thepoorly differentiated, moderately differentiated, andwell-differentiated forms.

This invention satisfies a need in the art for treatments that canremove benign tumors with less risk and fewer of the undesirable sideeffects of surgery. A method for removing benign tumors in surgicallyhazardous areas such as in deep locations in the body (e.g., brain,heart, lungs, and others) is particularly needed.

The method of treating conditions where cells must be removed can beused in conjunction with conventional methods of treating suchconditions, such as surgical excision, chemotherapy, and radiation. NTPpeptides can be administered before, during, or after such conventionaltreatments.

The condition to be treated can also be a hyperplasia, hypertrophy, orovergrowth of a tissue selected from the group consisting of lung,breast, stomach, pancreas, prostate, bladder, bone, ovary, skin, kidney,sinus, colon, intestine, stomach, rectum, esophagus, blood, brain andits coverings, spinal cord and its coverings, muscle, connective tissue,adrenal, parathyroid, thyroid, uterus, testis, pituitary, reproductiveorgans, liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, andlymph nodes and lymphoid system.

Other conditions that can be treated using the method of the inventionare virally, bacterially, or parasitically altered tissue selected fromthe group consisting of lung, breast, stomach, pancreas, prostate,bladder, bone, ovary, skin, kidney, sinus, colon, intestine, stomach,rectum, esophagus, blood, brain and its coverings, spinal cord and itscoverings, muscle, connective tissue, adrenal, parathyroid, thyroid,uterus, testis, pituitary, reproductive organs, liver, gall bladder,eye, ear, nose, throat, tonsils, mouth, and lymph nodes and lymphoidsystem.

The condition to be treated can also be a malformation or disorder of atissue selected from the group consisting of lung, breast, stomach,pancreas, prostate, bladder, bone, ovary, skin, kidney, sinus, colon,intestine, stomach, rectum, esophagus, blood, brain and its coverings,spinal cord and its coverings, muscle, connective tissue, adrenal,parathyroid, thyroid, uterus, testis, pituitary, reproductive organs,liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, and lymphnodes and lymphoid system.

In particular, the condition to be treated can be tonsillar hypertrophy,prostatic hyperplasia, psoriasis, eczema, dermatoses or hemorrhoids. Thecondition to be treated can be a vascular disease, such asatherosclerosis or arteriosclerosis, or a vascular disorder, such asvaricose veins, stenosis or restenosis of an artery or a stent. Thecondition to be treated can also be a cosmetic modification to a tissue,such as skin, eye, ear, nose, throat, mouth, muscle, connective tissue,hair, or breast tissue.

Therapeutic compositions of NTP peptides also are contemplated in thepresent invention. Such compositions may comprise a therapeuticallyeffective amount of an NTP peptide in admixture with a pharmaceuticallyacceptable carrier. The carrier material may be water for injection,preferably supplemented with other materials common in solutions foradministration to mammals. Typically, an NTP peptide for therapeutic usewill be administered in the form of a composition comprising purifiedNTP peptide in conjunction with one or more physiologically acceptablecarriers, excipients, or diluents. Neutral buffered saline or salinemixed with serum albumin are exemplary appropriate carriers. Preferably,the product is formulated as a lyophilizate using appropriate excipients(e.g., sucrose). Other standard carriers, diluents, and excipients maybe included as desired. Compositions of the invention also may comprisebuffers known to those having ordinary skill in the art with anappropriate range of pH values, including Tris buffer of about pH7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may furtherinclude sorbitol or a suitable substitute therefor.

The use of NTP peptides conjugated or linked or bound to an antibody,antibody fragment, antibody-like molecule, or a molecule with a highaffinity to a specific tumor marker, such as a cellular receptor, signalpeptide or over-expressed enzyme, for targeting to the unwanted cellularelements also is encompassed by the scope of the invention. Theantibody, antibody fragment, antibody-like molecule, or molecule with ahigh affinity to a specific tumor marker is used to target the NTPpeptide conjugate to a specific cellular or tissue target. For example,a tumor with a distinctive surface antigen or expressed antigen can betargeted by the antibody, antibody fragment, or antibody-like bindingmolecule and the tumor cells can be killed by the NTP peptide. Such anapproach using antibody targeting has the anticipated advantages ofdecreasing dosage, increasing the likelihood of binding to and uptake bythe target cells, and increased usefulness for targeting and treatingmetastatic tumors and microscopic sized tumors.

This invention also encompasses the use of NTP peptides conjugated orlinked or bound to a protein or other molecule to form a compositionthat, upon cleavage at or near the site(s) of the tumor or otherunwanted cells by a tumor- or site-specific enzyme or protease or by anantibody conjugate that targets tumor or other unwanted cells, releasesthe NTP peptide at or near the site(s) of the tumor or other unwantedcells

This invention also encompasses the use of NTP peptides conjugated orlinked or bound to a protein or other molecule to form a compositionthat releases the NTP peptide or some biologically active fragment ofthe NTP peptide upon exposure of the tissue to be treated to light (asin laser therapies or other photo-dynamic or photo-activated therapy),other forms of electro-magnetic radiation such as infra-red radiation,ultraviolet radiation, x-ray or gamma ray radiation, localized heat,alpha or beta radiation, ultrasonic emissions, or other sources oflocalized energy.

The NTP peptides may be employed alone, together, or in combination withother pharmaceutical compositions, such as cytokines, growth factors,antibiotics, apoptotis-inducing agents, anti-inflammatories, and/orchemotherapeutic agents as is appropriate for the indication beingtreated.

This invention also encompasses therapeutic compositions of NTP peptidesemploying dendrimers, fullerenes, and other synthetic molecules,polymers and macromolecules where the NTP peptide and/or itscorresponding DNA molecule is conjugated with, attached to or enclosedin the molecule, polymer or macromolecule, either by itself or inconjunction with other species of molecule such as a tumor-specificmarker. For example, U.S. Pat. No. 5,714,166, Bioactive and/or TargetedDendimer Conjugates, provides a method of preparing and using, intercilia, dendritic polymer conjugates composed of at least one dendrimerwith a target director(s) and at least one bioactive agent conjugated toit. The disclosure of U.S. Pat. No. 5,714,166 is incorporated byreference herein in its entirety.

This invention also encompasses therapeutic compositions of NTP peptidesand/or genes and drug delivery vehicles such as lipid emulsions, micellepolymers, polymer microspheres, electroactive polymers, hydrogels andliposomes.

The use of NTP peptides or related genes or gene equivalents transferredto the unwanted cells also is encompassed by the invention.Overexpression of NTP peptide within the tumor can be used to induce thecells in the tumor to die and thus reduce the tumor cell population. Thegene or gene equivalent transfer of NTP peptide to treat the unwantedcellular elements is anticipated to have the advantage of requiring lessdosage, and of being passed on to the cellular progeny of the targetedcellular elements, thus necessitating less frequent therapy, and lesstotal therapy. This invention also encompasses the transfer of genesthat code for a fusion protein containing an NTP peptide to the unwantedcells or neighboring cells where, following the expression of the geneand the production and/or secretion of the fusion protein, the fusionprotein is cleaved either by native enzymes or proteases or by a prodrugto release the NTP peptide in, at or near the unwanted cells.

The use of cloned recombinant NTP peptide-antibody conjugates; clonedrecombinant NTP peptide-antibody fragment conjugates; and clonedrecombinant NTP peptide-antibody-like protein conjugates is alsoencompassed by the scope of the invention. The advantages of a clonedNTP peptide combined with targeting conjugate (such as an antibody,antibody fragment, antibody-like molecule, or a molecule with a highaffinity to a cancer-specific receptor or other tumor marker) are thatsuch a molecule combines the targeting advantages described above inaddition to advantages for manufacturing and standardized production ofthe cloned conjugated molecule.

This invention also encompasses the use of therapeutic compositions ofNTP peptides or NTP genes or gene equivalents as a component of thecoating of a medical device such as a stent in order to remove, inhibitor prevent unwanted cellular proliferation or accumulation.

Solid dosage forms for oral administration include but are not limitedto, capsules, tablets, pills, powders, and granules. In such soliddosage forms, the active compound is admixed with at least one of thefollowing: (a) one or more inert excipients (or carrier), such as sodiumcitrate or dicalcium phosphate; (b) fillers or extenders, such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid; (c)binders, such as carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose and acacia; (d) humectants, such asglycerol; (e) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain complexsilicates, and sodium carbonate; (f) solution retarders, such asparaffin; (g) absorption accelerators, such as quaternary ammoniumcompounds; (h) wetting agents, such as acetyl alcohol and glycerolmonostearate; (i) adsorbents, such as kaolin and bentonite; and (j)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, or mixtures thereof. Forcapsules, tablets, and pills, the dosage forms may also comprisebuffering agents.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may compriseinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents, and emulsifiers. Exemplary emulsifiersare ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Actual dosage levels of active ingredients in the compositions of theinvention may be varied to obtain an amount of NTP peptide that iseffective to obtain a desired therapeutic response for a particularcomposition and method of administration. The selected dosage leveltherefore depends upon the desired therapeutic effect, the route ofadministration, the desired duration of treatment, and other factors.

With mammals, including humans, the effective amounts can beadministered on the basis of body surface area. The interrelationship ofdosages for animals of various sizes, species and humans (based on mg/m²of body surface) is described by E. J. Freireich et al., CancerChemother. Rep., 50 (4):219 (1966). Body surface area may beapproximately determined from the height and weight of an individual(see e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y. pp.537-538 (1970)).

The total daily dose of the NTP peptide administered to a host may be insingle or divided doses. Dosage unit compositions may contain suchamounts of such submultiples thereof as may be used to make up the dailydose. It will be understood, however, that the specific dose level forany particular patient will depend upon a variety of factors includingthe body weight, general health, sex, diet, time and route ofadministration, potency of the administered drug, rates of absorptionand excretion, combination with other drugs and the severity of theparticular disease being treated.

A method of administering an NTP peptide composition according to theinvention includes, but is not limited to, administering the compoundsintramuscularly, orally, intravenously, intraperitoneally,intracerebrally (intraparenchymally), intracerebroventricularly,intratumorally, intralesionally, intradermally, intrathecally,intranasally, intraocularly, intraarterially, topically, transdermally,via an aerosol, infusion, bolus injection, implantation device,sustained release system etc.

Another method of administering an NTP peptide of the invention is by atransdermal or transcutaneous route. One example of such an embodimentis the use of a patch. In particular, a patch can be prepared with afine suspension of NTP peptide in, for example, dimethylsulfoxide(DMSO), or a mixture of DMSO with cottonseed oil and brought intocontact with the skin of the tumor carrying mammals away from the tumorlocation site inside a skin pouch. Other mediums or mixtures thereofwith other solvents and solid supports would work equally as well. Thepatch can contain the NTP peptide compound in the form of a solution ora suspension. The patch can then be applied to the skin of the patient,for example, by means of inserting it into a skin pouch of the patientformed by folding and holding the skin together by means of stitches,clips or other holding devices. This pouch should be employed in such amanner so that continuous contact with the skin is assured without theinterference of the mammal. Besides using a skin pouch, any device canbe used which ensures the firm placement of the patch in contact withthe skin. For instance, an adhesive bandage could be used to hold thepatch in place on the skin.

NTP peptide may be administered in a sustained release formulation orpreparation. Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers, 22: 547-556 [1983]), poly (2-hydroxyethyl-methacrylate)(Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981] and Langer,Chem. Tech., 12: 98-105 [1982]), ethylene vinyl acetate (Langer et al.,supra) or poly-D(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (e.g., Eppstein etal., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 [1985]; EP 36,676; EP88,046; and EP 143,949).

Another method of administering an NTP peptide of the invention is bydirect or indirect infusion of NTP peptide into the tumor or othertissue to be treated. One example of such an embodiment is the directinjection of NTP peptide into the tumor or other tissue to be treated.The treatment may consist of a single injection, multiple injections onone occasion or a series of injections over a period of hours, days ormonths with the regression or destruction of the tumor or other tissueto be treated being monitored by means of biopsy, imaging or othermethods of monitoring tissue growth. The injection into the tumor orother tissue to be treated may be by a device inserted into an orificesuch as the nose, mouth, ear, vagina, rectum or urethra or through anincision in order to reach the tumor or tissue in vivo and may performedin conjunction with an imaging or optical system such as ultrasound orfibre optic scope in order to identify the appropriate site for theinjection(s). Another example of such an embodiment is the use of adevice that can provide a constant infusion of NTP peptide to the tissueover time.

Another method of administering an NTP peptide of the invention is inconjunction with a surgical or similar procedure employed to physicallyexcise, ablate or otherwise kill or destroy tumor or other tissue orcellular elements required or desired to be removed or destroyed whereinan NTP peptide of the invention is administered to the immediate area(s)surrounding the area(s) where the tumor or other tissue was removed inorder to destroy or impede the growth of any tumor cells or othercellular elements not removed or destroyed by the procedure

Another method of administering an NTP peptide of the invention is byimplantation of a device within the tumor or other tissue to be treated.One example of such an embodiment is the implantation of a wafercontaining NTP peptide in the tumor or other tissue to be treated. Thewafer releases a therapeutic dose of NTP peptide into the tissue overtime. Alternatively or additionally, the composition may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which the NTP peptide has beenabsorbed. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the NTPpeptide may be directly through the device via bolus, or via continuousadministration, or via catheter using continuous infusion.

An alternative method of administration is to introduce one or morecopies of an NTP peptide-encoding gene into the cell being targeted and,if necessary, inducing the copy(ies) of the gene to begin producing NTPpeptide intracellularly. One manner in which gene therapy can be appliedis to use the NTP peptide-encoding gene (either genomic DNA, cDNA,and/or synthetic DNA encoding the NTP peptide (or a fragment, variant,homologue or derivative thereof)) which may be operably linked to aconstitutive or inducible promoter to form a gene therapy DNA construct.The promoter may be homologous or heterologous to an endogenous NTPpeptide-encoding gene, provided that it is active in the cell or tissuetype into which the construct will be inserted. Other components of thegene therapy DNA construct may optionally include, as required, DNAmolecules designed for site-specific integration (e.g., endogenousflanking sequences useful for homologous recombination), tissue-specificpromoter, enhancer(s) or silencer(s), DNA molecules capable of providinga selective advantage over the parent cell, DNA molecules useful aslabels to identify transformed cells, negative selection systems, cellspecific binding agents (as, for example, for cell targeting)cell-specific internalization factors, and transcription factors toenhance expression by a vector as well as factors to enable vectormanufacture.

Means of gene delivery to a cell or tissue in vivo or ex vivo include(but are not limited to) direct injection of bare DNA, ballisticmethods, liposome-mediated transfer, receptor-mediated transfer(ligand-DNA complex), electroporation, and calcium phosphateprecipitation. See U.S. Pat. No. 4,970,154, WO 96/40958, U.S. Pat. No.5,679,559, U.S. Pat. No. 5,676,954, and U.S. Pat. No. 5,593,875, thedisclosures of each of which are incorporated by reference herein intheir entirety. They also include use of a viral vector such as aretrovirus, adenovirus, adeno-associated virus, pox virus, lentivirus,papilloma virus or herpes simplex virus, use of a DNA-protein conjugateand use of a liposome. The use of gene therapy vectors is described, forexample, in U.S. Pat. No. 5,672,344, U.S. Pat. No. 5,399,346, U.S. Pat.No. 5,631,236, and U.S. Pat. No. 5,635,399, the disclosures of each ofwhich are incorporated by reference herein in their entirety.

The NTP peptide-encoding gene may be delivered through implanting intopatients certain cells that have been genetically engineered ex vivo,using methods such as those described herein, to express and secrete theNTP peptide or fragments, variants, homologues, or derivatives thereof.Such cells may be animal or human cells, and may be derived from thepatient's own tissue or from another source, either human or non-human.Optionally, the cells may be immortalized or be stem cells. However, inorder to decrease the chance of an immunological response, it ispreferred that the cells be encapsulated to avoid infiltration ofsurrounding tissues. The encapsulation materials are typicallybiocompatible, semi-permeable polymeric enclosures or membranes thatallow release of the protein product(s) but prevent destruction of thecells by the patient's immune system or by other detrimental factorsfrom the surrounding tissues. Methods used for membrane encapsulation ofcells are familiar to the skilled artisan, and preparation ofencapsulated cells and their implantation in patients may beaccomplished without undue experimentation. See, e.g., U.S. Pat. Nos.4,892,538; 5,011,472; and 5,106,627, the disclosures of each of whichare incorporated by reference herein in their entirety. A system forencapsulating living cells is described in PCT WO 91/10425. Techniquesfor formulating a variety of other sustained or controlled deliverymeans, such as liposome carriers, bio-erodible particles or beads, arealso known to those in the art, and are described, for example, in U.S.Pat. No. 5,653,975, the disclosure of which is incorporated by referenceherein in their entirety. The cells, with or without encapsulation, maybe implanted into suitable body tissues or organs of the patient.

The following examples are provided to illustrate the present invention.It should be understood, however, that the invention is not to belimited to the specific conditions or details described in theseexamples. Throughout the specification, any and all references to apublicly available document, including a U.S. patent, are specificallyincorporated by reference.

In particular, this invention expressly incorporates by reference theexamples contained in pending U.S. patent application Ser. No.10/092,934, Methods of Treating Tumors and Related Conditions UsingNeural Thread Proteins, which reveal that the whole AD7c-NTP protein isan effective agent for causing cell death both in vitro in glioma andneuroblastoma cell cultures and in vivo in normal rodent muscle tissue,subcutaneous connective tissue, and dermis and in a variety of differenthuman and non-human origin tumors, including mammary carcinoma, skincarcinoma and papilloma, colon carcinoma, glioma of brain, and others inrodent models. This invention also expressly incorporates by referencethe examples contained in pending U.S. patent application Ser. No.10/153,334, entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells; Ser. No.10/198,069, entitled: Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells; and Ser.No. 10/198,070, entitled: Peptides Effective In The Treatment Of TumorsAnd Other Conditions Requiring The Removal Or Destruction Of Cells, eachof which reveal that fragments of AD7c-NTP, of proteins homologous toAD7c-NTP and NTP proteins, and of NTP proteins are effective agents forcausing cell death in vivo in normal rodent muscle tissue, subcutaneousconnective tissue, dermis and other tissue.

Example 1

The purpose of this example was to determine the effect of NTP[122]peptide #1 on tissue at sites of injection.

Male Sprague-Dawley rats (300 gram weight range) were anesthetized withether and given NTP[122] peptide #1 by intraprostatic infusion afteropen surgical visualization of the prostate. The injections consisted of300 μl of NTP[122] peptide #1, 1 mg/mL in PBS pH 7.4. (1.0 mg/kg) (n=8),control injections of PBS alone (n=6), and controls with no injection(n=2). Rats were painlessly sacrificed after 72 hours. Prostate glandswere dissected, fixed in 10% buffered formalin for 24 hours, embedded inparaffin, sectioned, and stained with H & E. For each animal the entireprostate gland was embedded and sectioned. All stained sections wereexamined histologically and measured. For each prostate at least 4histological sections were examined, and for each histological sectiontwo cross-sectional diameters (D) at 90° from each other were measured(total of ≧8 measurements per prostate). The mean diameter from thesemeasurements for each prostate was used to estimate volume according to

$V = {\frac{4}{3}{\left( \frac{D}{2} \right)^{3}.}}$

Results:

The reduction in prostate volume in NTP[122] peptide #1 injected ratswas estimated to be on average 45% compared to controls (there was nodiscernible difference between control PBS injections alone, andcontrols with no injections). Treated rat prostate showed extensive lossof glandular epithelium, flattening and atrophy. NTP[122] peptide #1 inPBS pH 7.4 open infusions of 1.0 mg/kg into rat prostate produced anestimated prostate volume reduction of >40% compared to untreated or PBStreated controls, at 72 hours.

Example 2

The purpose of this example was to determine the effect of NTP[112]peptide #1 on tissue at sites of injection.

Male Sprague-Dawley rats (300 gram weight range) were anesthetized withether and given NTP[112] peptide #1 by intraprostatic infusion afteropen surgical visualization of the prostate. The injections consisted of300 μl of NTP[112] peptide #1, 1 mg/mL in PBS pH 7.4. (1.0 mg/kg) (n=4),control injections of PBS alone (n=3), and controls with no injection(n=1). Rats were painlessly sacrificed after 72 hours. Prostate glandswere dissected, fixed in 10% buffered formalin for 24 hours, embedded inparaffin, sectioned, and stained with H & E. For each animal the entireprostate gland was embedded and sectioned. All stained sections wereexamined histologically and measured. For each prostate at least 4histological sections were examined, and for each histological sectiontwo cross-sectional diameters (D) at 90° from each other were measured(total of ≧8 measurements per prostate). The mean diameter from thesemeasurements for each prostate was used to estimate volume according to

$V = {\frac{4}{3}{\left( \frac{D}{2} \right)^{3}.}}$

The controls were the same as Example 1.

Results:

As in the above Example 1, injection of NTP[112] peptide #1 producedsignificant cell loss and atrophy in the prostate at 72 hours. Controlsshowed minimal or absent changes, consisting of mild focal inflammationfrom the needles.

Example 3

The purpose of this example was to determine the effect of the abovedescribed NTP peptides on tissue at sites of injection.

Eight normal rats were injected in the skin and subcutaneously, each infour different foci, and in extremity skeletal muscle, each in twodifferent foci, with the NTP[122] peptides 1-8, NTP[112] peptides 1-7,NTP[106] peptides 1-7, NTP[98] 1-6, NTP[75] peptides 1-5, NTP[68]peptides 1-4 and NTP[61] peptides 1-4 described above in saline inquantities of 100 to 400 mL at concentrations of 0.1-1 mg/mL deliveredfrom plastic syringes through stainless steel 26 gauge needles.

The animals were observed for 24 hours and painlessly sacrificed at 24hours. The individual foci of infiltration were excised, fixed in 10%formalin, embedded in paraffin, and stained and examined by standardhistopathological methods.

Similar groups of control rats were injected with (1) bovine serumalbumin 0.1% in saline, (2) normal human serum, (3) physiologicalsaline, (4) noninfectious bacterial proteins, and (5) control peptidesand purified and then examined and sacrificed as above, with the excisedfoci of injection treated as above.

Results

Injection of the NTP[122] peptides 1-8, NTP[112] peptides 1-7, NTP[106]peptides 1-7, NTP[98] 1-6, NTP[75] peptides 1-5, NTP[68] peptides 1-4and NTP[61] peptides 1-4 in all examples produced abundant acutenecrosis of tissue at the injection sites. The necrosis is evident inmuscle tissue, subcutaneous connective tissue, and dermis at the siteswhere the NTP peptide was injected. At 24 hours, cells appear pale,shrunken, and necrotic, and there is infiltration with inflammatorycells. The necrosis correlates with the areas of injection and does notappear to spread far beyond the site of injection.

Apart from the mild areas of inflammation, controls showed no evidenceof necrosis or cell loss. In contrast to the NTP peptide injectionswhere entire fields of muscle fiber layers were necrotic, the controlsshowed minimal or absent muscle changes. Control injections had mild tominimal acute inflammation at the injection sites and focalmicrohemorrhages from the needles.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention.

What is claimed is:
 1. An isolated NTP peptide consisting of SEQ ID NO.8 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu)in the form of a salt.
 2. The isolated NTP peptide of claim 1, whereinthe isolated peptide is chemically synthesized.
 3. A compositioncomprising the isolated NTP peptide according to claim 1, and a carriertherefor.
 4. An isolated NTP peptide consisting of SEQ ID NO. 8(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu),modified by from 1 to 25 additional amino acids flanking the aminoand/or carboxy termini of the peptide.
 5. An isolated NTP peptideconsisting of at least two repetitions of the NTP peptide according toclaim
 1. 6. An isolated NTP peptide consisting of the NTP peptide ofclaim 1, fused to an antibody or antigen binding fragment of anantibody.